Recombinant alphavirus-based vectors with reduced inhibition of cellular macromolecular synthesis

ABSTRACT

Isolated nucleic acid molecules are disclosed, comprising an alphavirus nonstructural protein gene which, when operably incorporated into a recombinant alphavirus particle, eukaryotic layered vector initiation system, or RNA vector replicon, has a reduced level of vector-specific RNA synthesis, as compared to wild-type, and the same or greater level of proteins encoded by RNA transcribed from the viral junction region promoter, as is compared to a wild-type recombinant alphavirus particle. Also disclosed are RNA vector replicons, alphavirus vector constructs, and eukaryotic layered vector initiation systems which contain the above-identified nucleic acid molecules.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a divisional of copending U.S. patent applicationSer. No. 08/944,465, filed Oct. 6,1997, which application is acontinuation-in-part of U.S. patent application Ser. No. 08/833,148,filed Apr. 4,1997, abandoned; which is a continuation-in-part of U.S.patent application Ser. No. 08/679,640, filed Jul. 12, 1996, abandoned;which is a continuation-in-part of U.S. patent application Ser. No.08/668,953 filed Jun. 24, 1996, abandoned, which is acontinuation-in-part of U.S. patent application Ser. No. 08/628,594,filed Apr. 5, 1996, abandoned, all of which are incorporated herein intheir entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention has been made in part with government support under grantnumber AI 11377, awarded by the National Institutes of Health. Thegovernment may have certain rights in the invention.

TECHNICAL FIELD OF THE INVENTION

The present invention relates generally to recombinant DNA technology;and more specifically, to the development of recombinant vectors usefulfor directing the expression of one or more heterologous gene products.

BACKGROUND OF THE INVENTION

Alphaviruses comprise a set of genetically, structurally, andserologically related arthropod-borne viruses of the Togaviridae family.These viruses are distributed worldwide, and persist in nature through amosquito to vertebrate cycle. Birds, rodents, horses, primates, andhumans are among the defined alphavirus vertebrate reservoir/hosts.

Twenty-six known viruses and virus subtypes have been classified withinthe alphavirus genus utilizing the hemagglutination inhibition (HI)assay. This assay segregates the 26 alphaviruses into three majorcomplexes: the Venezuelan equine encephalitis (VEE) complex, the SemlikiForest (SF) complex, and the western equine encephalitis (WEE) complex.In addition, four other viruses, eastern equine encephalitis (EEE),Barmah Forest, Middelburg, and Ndumu, receive individual classificationbased on the HI serological assay.

Members of the alphavirus genus also are classified based on theirrelative clinical features in humans: alphaviruses associated primarilywith encephalitis, and alphaviruses associated primarily with fever,rash, and polyarthritis. Included in the former group are the VEE andWEE complexes, and EEE. In general, infection with this group can resultin permanent sequelae, including behavior changes and learningdisabilities, or death. In the latter group is the SF complex, comprisedof the individual alphaviruses Semliki Forest, Sindbis, Ross River,Chikungunya, O'nyong-nyong, and Mayaro. With respect to this group,although serious epidemics have been reported, infection is in generalself-limiting, without permanent sequelae.

Sindbis virus is the prototype member of the Alphavirus genus of theTogaviridae family. Its replication strategy after infection of cells(see FIG. 1) has been well characterized in chicken embryo fibroblasts(CEF) and baby hamster kidney (BHK) cells, where Sindbis virus growsrapidly and to high titer, and serves as a model for other alphaviruses.Briefly, the genome from Sindbis virus (like other alphaviruses) is anapproximately 12 kb single-stranded positive-sense RNA molecule which iscapped and polyadenylated, and contained within a virus-encoded capsidprotein shell. The nucleocapsid is further surrounded by a host-derivedlipid envelope into which two viral-specific glycoproteins, E1 and E2,are inserted and anchored to the nucleocapsid. Certain alphaviruses(e.g., SF) also maintain an additional protein, E3, which is a cleavageproduct of the E2 precursor protein, PE2. After virus particleabsorption to target cells, penetration, and uncoating of thenucleocapsid to release viral genomic RNA into the cytoplasm, thereplicative process is initiated by translation of the nonstructuralproteins (nsPs) from the 5′ two-thirds of the viral genome. The fournsPs (nsP1-nsP4) are translated directly from the genomic RNA templateas one of two polyproteins (nsP123 or nsP1234), and processedpost-translationally into monomeric units by an active protease in theC-terminal domain nsP2. A leaky opal (UGA) codon present between nsP3and nsP4 of most alphaviruses accounts for a 10 to 20% abundance of thensP1234 polyprotein, as compared to the nsP123 polyprotein. Both of thenonstructural polyproteins and their derived monomeric units mayparticipate in the RNA replicative process, which involves binding tothe conserved nucleotide sequence elements (CSEs) present at the 5′ and3′ ends, and a junction region subgenomic promoter located internally inthe genome (discussed further below).

The positive strand genomic RNA serves as template for the nsP-catalyzedsynthesis of a full-length complementary negative strand. Synthesis ofthe complementary negative strand is catalyzed after binding of the nsPcomplex to the 3′ terminal CSE of the positive strand genomic RNA. Thenegative strand, in turn, serves as template for the synthesis ofadditional positive strand genomic RNA and an abundantly expressed 26Ssubgenomic RNA, initiated internally at the junction region promoter.Synthesis of additional positive strand genomic RNA occurs after bindingof the nsP complex to the 3′ terminal CSE of the complementary negativestrand genomic RNA template. Synthesis of the subgenomic mRNA from thenegative strand genomic RNA template, is initiated from the junctionregion promoter. Thus, the 5′ end and junction region CSEs of thepositive strand genomic RNA are functional only after they aretranscribed into the negative strand genomic RNA complement (i.e., the5′ end CSE is functional when it is the 3′ end of the genomic negativestranded complement). The structural proteins (sPs) are translated fromthe subgenomic 26S RNA, which represents the 3′ one-third of the genome,and like the nsPs, are processed post-translationally into theindividual proteins.

Several groups have suggested utilizing certain members of thealphavirus genus as an expression vector, including, for example,Sindbis virus (Xiong et al., Science 243:1188-1191,1989; Hahn et al.,Proc. Natl. Acad. Sci. USA 89:2679-2683, 1992; Dubensky et al., J.Virol. 70:508-519,1996), Semliki Forest virus (Liljestrom,Bio/Technology 9:1356-1361,1991), and Venezuelan Equine Encephalitisvirus (Davis et al., J. Cell. Biochem. Suppl. 19A:10,1995). In addition,one group has suggested using alphavirus-derived vectors for thedelivery of therapeutic genes in vivo. One difficulty, however, with theabove-referenced vectors is that inhibition of host cell-directedmacromolecular synthesis (i.e., protein or RNA synthesis) begins withina few hours after infection and cytopathic effects (CPE) occur within 12to 16 hours post infection (hpi). Inhibition and shutoff of host cellprotein synthesis begins within 2 hpi in BHK cells infected withrecombinant viral particles, in the presence or absence of structuralprotein expression, suggesting that the early events after virusinfection (e.g., synthesis of nsPs and minus strand RNA) may directlyinfluence the inhibition of host cell protein synthesis and subsequentdevelopment of CPE and cell death.

SIN-1 is a variant strain derived from wild-type Sindbis, and wasisolated from a culture of BHK cells persistently infected with Sindbisvirus over a period of one month (Weiss et al. J. Virol. 33: 463-474.1980). A pure SIN-1 virus stock obtained by expansion from a singleplaque does not kill the BHK cells which it infects. Importantly, virusyields (>10³ PFU/cell) are the same in BHK cells infected with wild-typeSindbis virus or the variant SIN-1 virus. Thus, the principle phenotypeof SIN-1 in infected BHK cells is characterized by production ofwild-type levels of infectious virus in the absence of virus-inducedcell death.

The present invention provides recombinant vectors with selecteddesirable phenotypes for use in a variety of applications, including forexample, gene therapy and recombinant protein production, and furtherprovides other related advantages.

SUMMARY OF THE INVENTION

Briefly stated, the present invention provides RNA vector replicons,alphavirus vector constructs, eukaryotic layered vector initiationsystems and recombinant alphavirus particles which exhibit reduced,delayed, or no inhibition of cellular macromolecular synthesis (e.g.,protein or RNA synthesis), thereby permitting the use of these vectorsfor protein expression, gene therapy and the like, with reduced,delayed, or no development of CPE or cell death. Such vectors may beconstructed from a wide variety of alphaviruses (e.g., Semliki Forestvirus, Ross River virus, Venezuelan equine encephalitis virus or Sindbisvirus), and designed to express numerous heterologous sequences (e.g., asequence corresponding to protein, a sequence corresponding to antisenseRNA, a sequence corresponding to non-coding sense RNA, or a sequencecorresponding to ribozyme).

Within one aspect of the invention, isolated nucleic acid molecules areprovided comprising an altered alphavirus nonstructural protein genewhich, when operably incorporated into a recombinant alphavirus,increases the time required to reach 50% inhibition of host-celldirected macromolecular synthesis following expression in mammaliancells, as compared to a wild-type alphavirus. As utilized within thecontext of the present invention, “altered alphavirus nonstructuralprotein gene” refers to a gene which, when operably incorporated into analphavirus RNA vector replicon, recombinant alphavirus particle, oreukaryotic layered vector initiation system, produces the desiredphenotype (e.g., reduced, delayed or no inhibition of cellularmacromolecular synthesis). Such altered alphavirus nonstructural proteingenes will have one or more nucleotide substitutions, deletions, orinsertions, which alter the nucleotide sequence from that of thewild-type alphavirus gene. The gene may be derived either artificially(e.g., from directed selection procedures; see Example 2 below), or fromnaturally occurring viral variants (see Example 1 below). In addition,it should be understood that when the isolated nucleic acid molecules ofthe present invention are incorporated into an alphavirus RNA vectorreplicon, recombinant alphavirus particle, or eukaryotic layered vectorinitiation system as discussed above, that they may, within certainembodiments, substantially increase the time required to reach 50%inhibition of host-cell directed macromolecular synthesis, up to andincluding substantially no detectable inhibition of host-cell directedmacromolecular synthesis (over any period of time). Assays suitable fordetecting percent inhibition of host-cell directed macromolecularsynthesis include, for example, that described within Example 1.

Within other aspects of the invention, isolated nucleic acid moleculesare provided comprising an altered alphavirus nonstructural protein genewhich, when operably incorporated into a recombinant alphavirusparticle, eukaryotic layered vector initiation system, or RNA vectorreplicon, results in a reduced level (e.g., 2-fold, 5-fold, 10-fold,50-fold or more than 100-fold) of vector-specific RNA synthesis ascompared to the wild-type, and the same or greater level of proteinencoded by RNA transcribed from the viral junction region promoter, ascompared to a wild-type recombinant alphavirus particle, wild-typeeukaryotic layered vector initiation system, or wild-type RNA vectorreplicon. Representative assays for quantitating RNA levels include [³H]uridine incorporation as described in Example 1, or RNA accumulation asdetected by Northern Blot analysis (see Example 4). Representativeassays for quantitating protein levels include scanning densitometry(see Example 4) and various enzymatic assays (see Examples 3-5).

Within one embodiment of the above, the isolated nucleic acid moleculeencodes nonstructural protein 2 (nsP2). Within a further embodiment, theisolated nucleic acid molecule has a mutation in the LXPGG motiff ofnsP2.

Within another aspect of the invention, expression vectors are providedcomprising a promoter operably linked to one of the above-describednucleic acid molecules. Within one embodiment, the expression vectorfurther comprises a polyadenylation sequence or transcriptiontermination sequence 3′ to the nucleic acid molecule.

Within yet another aspect of the present invention, alphavirus vectorconstructs are provided, comprising a 5′ promoter which initiatessynthesis of viral RNA in vitro from cDNA, a 5′ sequence which initiatestranscription of alphavirus RNA, a nucleic acid molecule which operablyencodes all four alphaviral nonstructural proteins including an isolatednucleic acid molecule as described above, an alphavirus viral junctionregion promoter, an alphavirus RNA polymerase recognition sequence and a3′ polyadenylate tract.

Within a related aspect, such constructs further comprise a selectedheterologous sequence downstream of and operably linked to a viraljunction region. Within a related aspect, alphavirus vector constructsare provided comprising a 5′ promoter which initiates synthesis of viralRNA in vitro from cDNA, a 5 ′ sequence which initiates transcription ofalphavirus RNA, a nucleic acid molecule which operably encodes all fouralphavirus non-structural proteins, an alphavirus viral junction regionpromoter, an alphavirus RNA polymerase recognition sequence, and a 3′polyadenylate tract, wherein said in vitro synthesized RNA, uponpackaging into an alphavirus particle and introduction of the particleinto a mammalian host cell, increases the time required to reach 50%inhibition of host-cell directed macromolecular synthesis followingexpression in mammalian cells, as compared to a wild-type alphavirusparticle.

Within a further aspect, alphavirus vector constructs are providedcomprising a 5′ promoter which initiates synthesis of viral RNA in vitrofrom cDNA, a 5′ sequence which initiates transcription of alphavirusRNA, a nucleic acid molecule which operably encodes all four alphavirusnon-structural proteins, an alphavirus viral junction region promoter,an alphavirus RNA polymerase recognition sequence, and a 3′polyadenylate tract, wherein said in vitro synthesized RNA, uponpackaging into an alphavirus particle and introduction of the particleinto a mammalian host cell, has a reduced level of vector-specific RNAsynthesis as compared to wild-type alphavirus particle, and the same orgreater level of protein encoded by RNA transcribed from the viraljunction region promoter, as compared to a wild-type alphavirusparticle.

Within yet other aspects of the present invention, RNA vector repliconscapable of translation in a eukaryotic system are provided, comprising a5′ sequence which initiates transcription of alphavirus RNA, a nucleicacid molecule which operably encodes all four alphaviral nonstructuralproteins, including the isolated nucleic acid molecules discussed above,an alphavirus viral junction region, an alphavirus RNA polymeraserecognition sequence and a 3′ polyadenylate tract.

Within a related aspect, alphavirus RNA vector replicons capable oftranslation in a eukaryotic system are provided, comprising a 5′sequence which initiates transcription of alphavirus RNA, a nucleic acidmolecule which operably encodes all four alphaviral nonstructuralproteins, an alphavirus viral junction region promoter, an alphaviruspolymerase recognition sequence and a 3′ polyadenylate tract, whereinsaid alphavirus RNA, upon packaging into an alphavirus particle andintroduction of the particle into a mammalian host cell, increases thetime required to reach 50% inhibition of host-cell directedmacromolecular synthesis following expression in mammalian cells, ascompared to a wild-type alphavirus particle.

Within other aspects, alphavirus RNA vector replicons capable oftranslation in a eukaryotic system are provided comprising a 5′ sequencewhich initiates transcription of alphavirus RNA, a nucleic acid moleculewhich operably encodes all four alphaviral nonstructural proteins, analphavirus viral junction region promoter, an alphavirus polymeraserecognition sequence and a 3′ polyadenylate tract, wherein saidalphavirus RNA, upon packaging into an alphavirus particle andintroduction of the particle into a mammalian host cell, has a reducedlevel of vector-specific RNA synthesis as compared to wild-typealphavirus particle, and the same or greater level of protein encoded byRNA transcribed from the viral junction region promoter, as compared toa wild-type alphavirus particle.

Within another embodiment, such RNA vector replicons further comprise aselected heterologous sequence downstream of and operably linked to aviral junction region. Within further aspects of the invention, hostcells are provided which contain one of the RNA vector repliconsdescribed herein. Within additional aspects of the invention,pharmaceutical compositions are provided comprising RNA vector repliconsas described above and a pharmaceutically acceptable carrier or diluent.

Within other aspects of the invention, recombinant alphavirus particlesare provided, comprising one or more alphavirus structural proteins, alipid envelope, and an RNA vector replicon as described herein. Withinone embodiment, one or more of the alphavirus structural proteins arederived from a different alphavirus than the alphavirus from which theRNA vector replicon was derived. Within other embodiments, thealphavirus structural protein and lipid envelopes are derived fromdifferent species. Within further aspects, pharmaceutical compositionsare provided comprising a recombinant alphavirus particle as disclosedabove and a pharmaceutically acceptable carrier or diluent. Further,mammalian cells infected with such recombinant alphavirus particles arealso provided.

Within certain embodiments of the invention the above described vectorsor particles may further comprise a resistance marker which has beenfused, in-frame, with the heterologous sequence. Representative examplesof such resistance markers include hygromycin phosphotransferase andneomycin phosphotransferase.

Within other aspects of the present invention, methods are provided forselecting alphavirus or recombinant alphavirus vector variants whichexhibit the phenotype described herein of reduced, delayed, or, noinhibition of host cell directed macromolecular synthesis.Representative examples of such methods include the use of selectabledrug or antigenic markers and are provided in more detail below inExample 2.

Within other aspects of the present invention, Togavirus capsidparticles are provided which contain substantially no genomic (i.e.,wild-type virus genome) or RNA vector replicon nucleic acids.Representative examples of Togaviruses include, for example alphavirusesand rubiviruses (e.g., rubella). Within certain embodiments, the capsidparticles further comprise a lipid envelope containing one or morealphavirus glycoproteins. Within other embodiments, the capsid particlefurther comprises an alphavirus envelope (i.e., the lipid bilayer andthe glycoprotein complement). Within related aspects of the presentinvention, pharmaceutical compositions are provided comprising the abovenoted capsid particles (with or without a lipid bilayer (e.g., viralenvelope containing alphavirus glycoproteins)) along with apharmaceutically acceptable carrier or diluent. Within further aspects,such capsid particles (with or without a lipid bilayer (e.g., viralenvelope containing alphavirus glycoproteins)) or pharmaceuticalcompositions may be utilized as a vaccinating agent in order to inducean immune response against a desired togavirus.

Within further aspects of the invention, inducible promoters areprovided comprising a core RNA polymerase promoter sequence, an operablylinked nucleic acid sequence that directs the DNA binding of a proteinthat activates transcription from the core promoter sequence, and anoperably linked nucleic acid sequence that directs the DNA binding of aprotein that represses transcription from the core promoter sequence.Such promoters may be utilized in the gene delivery vehicles describedherein, as well as a wide variety of other vectors known to thoseskilled in the art.

Within other aspects, alphavirus structural protein expression cassettesare provided comprising a 5′ promoter which initiates synthesis of viralRNA from DNA, a nucleic acid molecule which encodes one or morefunctional alphavirus structural proteins, a selectable marker operablylinked to transcription of the expression cassette, and optionally, a 3′sequence which controls transcription termination. Within oneembodiment, such expression cassettes further comprise a 5′ sequencewhich initiates transcription of alphavirus RNA, a viral junction regionpromoter, and an alphavirus RNA polymerase recognition sequence. Withinanother embodiment, the expression cassette further comprises acatalytic ribozyme processing sequence, post-translationaltranscriptional regulatory elements which facilitate RNA export from thenucleus, and/or elements which permit translation of multicistronicMRNA, selected from the group consisting of Internal Ribosome Entry Siteelements, elements promoting ribosomal read through and BiP sequence.Within other embodiments, the selectable marker is operably linked to a5′ promoter capable of initiating synthesis of alphavirus RNA from cDNA.Within further embodiments the selectable marker is positioneddownstream from a junction region promoter and from the nucleic acidmolecule which encodes alphavirus structural proteins. Within yet otherembodiments, the 5′ promoter is an inducible promoter as describedherein. Within another embodiment, the alphavirus structural proteinexpression cassette further comprises an alphavirus capsid protein geneor other sequence (e.g., a tobacco etch virus or “TEV” leader) which iscapable of enhancing translation of one or more functional alphavirusstructural protein genes located 3′ to the enhancer sequence.Preferably, the capsid protein gene sequence is derived from a differentalphavirus than that from which the sequence encoding the alphavirusstructural genes is obtained.

Within yet other aspects of the invention, alphavirus packaging celllines are provided comprising a cell containing an alphavirus structuralprotein expression cassette as described above. In certain embodiments,the alphavirus packaging cell lines are stably transformed with thealphavirus structural protein expression cassettes provided herein.Within related aspects, alphavirus producer cell lines are providedcomprising a cell which contains a stably transformed alphavirusstructural protein expression cassette, and a vector selected from thegroup consisting of RNA vector replicons, alphavirus vector constructsand eukaryotic layered vector initiation systems.

Within yet other aspects of the present invention, eukaryotic layeredvector initiation systems are provided, comprising a 5′ promoter capableof initiating in vivo the 5′ synthesis of RNA from cDNA, a sequencewhich initiates transcription of alphavirus RNA following the 5′promoter; a nucleic acid molecule which operably encodes all fouralphaviral nonstructural proteins, including an isolated nucleic acidmolecule as discussed above, an alphavirus RNA polymerase recognitionsequence, and a 3′ polyadenylate tract.

Also provided are eukaryotic layered vector initiation systemscomprising a 5′ promoter capable of initiating in vivo the 5′ synthesisof alphavirus RNA from cDNA, a sequence which initiates transcription ofalphavirus RNA following the 5′ promoter, a nucleic acid molecule whichoperably encodes all four alphaviral nonstructural proteins, analphavirus RNA polymerase recognition sequence, and a 3′ polyadenylatetract, wherein the in vivo synthesized RNA, upon packaging into analphavirus particle and introduction of the particle into a mammalianhost cell, increases the time required to reach 50% inhibition ofhost-cell directed macromolecular synthesis following expression inmammalian cells, as compared to a wild-type alphavirus particle.

Related eukaryotic layered vector initiation system are also providedwhich comprise a 5′ promoter capable of initiating in vivo the 5′synthesis of alphavirus RNA from cDNA, a sequence which initiatestranscription of alphavirus RNA following the 5′ promoter, a nucleicacid molecule which operably encodes all four alphaviral nonstructuralproteins, an alphavirus RNA polymerase recognition sequence, and a 3′polyadenylate tract, wherein said in vivo synthesized RNA, uponpackaging into an alphavirus particle and introduction of the particleinto a mammalian host cell, has a reduced level of vector-specific RNAsynthesis as compared to wild-type alphavirus particle, and the same orgreater level of protein encoded by RNA transcribed from the viraljunction region promoter, as compared to a wild-type alphavirusparticle.

Representative examples of suitable 5′ promoters for eukaryotic layeredvector initiation systems include RNA polymerase I promoters, RNApolymerase II promoters, RNA polymerase III promoters, the HSV-TKpromoter, RSV promoter, tetracycline inducible promoter, MoMLV promoter,a SV40 promoter and a CMV promoter. Within preferred embodiments, the 5′promoter is an inducible promoter as described herein.

Within certain embodiments, eukaryotic layered vector initiation systemsare provided which further comprise a heterologous sequence operablylinked to a viral junction region, and/or a post-transcriptionalregulatory element which facilitates RNA export from the nucleus. Withinfurther embodiments, the eukaryotic layered vector initiation systemsprovided herein may further comprise a transcription termination signal.

Within related aspects, the present invention also provides host cells(e.g., vertebrate or insect) containing a stably transformed eukaryoticlayered vector initiation system as described above. Within furtheraspects of the present invention, methods for delivering a selectedheterologous sequence to a vertebrate or insect are provided, comprisingthe step of administering to a vertebrate or insect an alphavirus vectorconstruct, RNA vector replicon, recombinant alphavirus particle, or aeukaryotic layered vector initiation system as described herein. Withincertain embodiments, the alphavirus vector construct, RNA vectorreplicon, recombinant alphavirus particle or eukaryotic layered vectorinitiation system is administered to cells of the vertebrate ex vivo,followed by administration of the vector or particle-containing cells toa warm-blooded animal.

Within other aspects, pharmaceutical compositions are providedcomprising a eukaryotic layered vector initiation system as discussedabove, and a pharmaceutically acceptable carrier or diluent. Withincertain embodiments, the pharmaceutical composition is provided as aliposomal formulation.

Within further aspects, methods of making recombinant alphavirusparticles are provided, comprising the steps of (a) introducing a vectorsuch as a eukaryotic layered vector initiation system, RNA vectorreplicon, or alphavirus vector particle as described above into apopulation of packaging cells under conditions and for a time sufficientto permit production of recombinant alphavirus particles, and (b)harvesting recombinant alphavirus particles. Within related aspects,methods of making a selected protein are provided, comprising the stepsof (a) introducing a vector which encodes a selected heterologousprotein, such as a eukaryotic layered vector initiation system, RNAvector replicon or alphavirus vector particle described above, into apopulation of packaging cells, or other cells under conditions and for atime sufficient to permit production of the selected protein, and (b)harvesting protein produced by the vector containing cells. Within yetother aspects, methods of making a selected protein are provided,comprising the step of introducing a eukaryotic layered vectorinitiation system which is capable of producing a selected heterologousprotein into a host cell, under conditions and for a time sufficient topermit expression of the selected protein. Within further aspects, hostcell lines are provided which contain a RNA vector replicon as describedherein.

Within yet other aspects of the present invention, alphavirus vaccinesare provided, comprising one of the above-described alphavirus vectorconstructs, RNA vector replicons, eukaryotic vector initiation systems,or recombinant alphavirus particles, which may or may not express one ofthe heterologous sequences provided herein (e.g., they may be utilizedsolely as a vaccine for treating or preventing alphaviral diseases). Forexample, within one embodiment of the invention, recombinant togavirusparticles are provided which have substantially no nucleic acid or RNAvector replicon nucleic acid. Within a further embodiment, recombinanttogavirus particles are provided which contain heterologous viralnucleic acids (ie., from a different virus than the togavirus particle).Within yet another embodiment, the recombinant togavirus particle is T=3or greater.

Within further aspects of the invention, recombinant chimeric togavirusparticles (either empty, or containing nucleic acids) are providedwherein the viral particle has viral structural components obtained orderived from different Togaviridae (e.g., the capsid protein andglycoprotein is obtained from different alphavirus sources).

Within other aspects of the invention, methods for stimulating an immuneresponse within a vertebrate are provided, comprising the step ofadministering to a vertebrate an alphavirus vector construct, analphavirus RNA vector replicon according, a recombinant alphavirusparticle, or a eukaryotic layered vector initiation system, wherein thealphavirus vector construct, RNA vector replicon, particle, oreukaryotic layered vector initiation system expresses an antigen whichstimulates an immune response within said vertebrate (see, e.g., U.S.Ser. No. 08/404, 796 for suitable antigens). Within related aspects,methods are provided for inhibiting a pathogenic agent within avertebrate, comprising the step of administering to a vertebrate analphavirus vector construct, an alphavirus RNA vector replicon, arecombinant alphavirus particle, or a eukaryotic layered vectorinitiation system according, wherein said alphavirus vector construct,RNA vector replicon, particle, or eukaryotic layered vector initiationsystem expresses an palliative which is capable of inhibiting apathogenic agent (see. e.g., U.S. Ser. No. 08/404, 796 for suitablepalliatives).

These and other aspects and embodiments of the invention will becomeevident upon reference to the following detailed description andattached figures. In addition, various references are set forth hereinthat describe in more detail certain procedures or compositions (e.g.plasmids, sequences, etc.), and are therefore incorporated by referencein their entirety as if each were individually noted for incorporation.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic illustration of Sindbis virus and generalalphavirus genomic organization and replication strategy.

FIG. 2 is a graph of virus release from BHK cells infected at an MOI of10 with SIN-1, SIN-1/nsP1-4, Toto1101, or Sin-1/nsP2 viruses. Cellculture fluids were collected at 3, 6, 9 and 12 hours post-infection.Virus titers were determined by plaque assay.

FIG. 3 is a graph depicting viral RNA synthesis in BHK cells followinginfection by Toto1101, SIN-1/nsP2, SIN-1/nsP1-4, or SIN-1 virus. Cellswere infected at an MOI 10 and at 1 hour post-infection, actinomycin Dand ³H-uridine were added. At 3, 6, 9, and 12 hpi the amount of³H-uridine incorporation was determined.

FIG. 4 is a graph depicting viral RNA synthesis in BHK cells infected bySIN-1/nsP1, SIN-1/nsP2, SIN-1/nsP3, SIN-1/nsP3-4, SIN-1/nsP4,SIN-1/nsP2-C, SIN-1/nsP2-N, Toto1101, SIN-1, or SIN-1/nsP1-4. The levelsof 3H-uridine incorporation are expressed relative to wild-type (Toto1101) infection.

FIG. 5 is a graph depicting the shut-off of host cell protein synthesisin BHK cells infected by SIN-InsP1-4, SIN-1, SIN-1nsP2, or Toto1101viruses.

FIGS. 6A-6D is the cDNA sequence of 8000 bases of SIN-1 virus (SEQ. IDNO. 101).

FIGS. 7A-7D is the cDNA sequence of 8000 bases of SINCG virus (SEQ. IDNO. 102).

FIGS. 8A-8E are the cDNA sequence of Toto 1101 virus (SEQ. ID NO. 103).

FIG. 8F is a schematic illustration depicting selection of vectorsexpressing the desired phenotype using a selectable marker.

FIG. 8G is a northern blot analysis of RNAs isolated from G418-resistantBHK-21 cell pools stably transformed with a variant Sindbis virus vectoror Semliki Forest virus vector expressing neomycin phosphotransferase.

FIG. 8H is a schematic illustration of the genetic determinantsresponsible for the desired phenotype in variant Sindbis virus vectors.

FIG. 9A is a northern blot analysis of RNAs isolated from BHK-21 cellsthat were transfected with pBG/SIN-1 ELVS 1.5-SEAP or pBG/wt ELVS1.5-SEAP plasmid DNAs, and hybridized with a radiolabeled viral RNAprobe.

FIG. 9B is a graph depicting a 7 day timecourse of alkaline phosphataseexpression in BHK cells transfected with pBG/SIN-1 ELVS 1.5-SEAP orpBG/wt ELVS 1.5-SEAP plasmid DNAs.

FIG. 10 is a graph depicting a 4 day timecourse of luciferase expressionin BHK, cells transfected with pBG/SIN-1 ELVS 1.5-luc or pBG/wt ELVS1.5-luc plasmid DNAs.

FIG. 11A is a northern blot analysis of RNAs isolated from BHK-21 cellsthat were transfected with pBG/SIN-1 ELVS-1.5-β-gal or pBG/wt ELVS1.5-β-gal plasmid DNAs, and hybridized with a radiolabeled viral RNAprobe.

FIG. 11B is a western blot analysis detecting β-gal expression in BHK-21cells transfected with either pBG/SIN-1 ELVS-1.5-β-gal or pBG/wt ELVS1.5-β-gal plasmid DNAs.

FIG. 11C is a graph depicting a 5 day timecourse of alkaline phosphataseexpression in BHK cells transfected with pBG/SIN-1 ELVS-1.5-β-gal orpBG/wt ELVS 1.5-β-gal plasmid DNAs.

FIGS. 12A & B are graphs depicting β-gal expression in HT1080 and BHK-21cells transfected with ELVS β-gal vectors with or without HBV PREsequences, as measured by RLU (relative light units).

FIG. 13 is a schematic illustration of RNA amplification, structuralprotein expression, and vector packaging by vector inducible alphaviruspackaging cell lines.

FIG. 14 is a schematic illustration of vector inducible structuralprotein expression cassettes used in the generation of alphaviruspackaging cell lines.

FIG. 15 is a graph depicting luciferase vector packaging (transfer ofexpression) by different alphavirus packaging cell lines.

FIG. 16 is a western blot analysis demonstrating induction of structuralprotein expression by an alphavirus packaging cell line followingtransfection and subsequent expression with an alphavirus vector(ELVS-βgal), but not a conventional plasmid DNA expression vector(pCMV-βgal).

FIG. 17A is a graph depicting luciferase vector packaging by C6/36mosquito cells containing the pDCMV-intSINrbz structural proteinexpression cassette.

FIG. 17B is a graph depicting luciferase vector packaging by human 293packaging cells stably transformed with plasmid pBGSVCMVdlneo.

FIG. 18 is a graph depicting luciferase vector packaging by differentalphavirus packaging cell lines.

FIG. 19 is an RNA gel autoradiograph depicting ³H uridine-labeled RNAsfrom BHK cells infected with SINrep/LacZ vector particles produced froman alphavirus packaging cell line.

FIG. 20 is a protein gel autoradiograph depicting ³⁵S methionine-labeledproteins from BHK cells infected with SINrep/LacZ vector particlesproduced from an alphavirus packaging cell line.

FIG. 21A is a schematic illustration depicting packaging of alphavirusvectors with structural proteins in which the capsid protein andglycoproteins are expressed from distinct, or “split”, expressioncassettes.

FIG. 21B is a schematic illustration depicting the structural proteinexpression cassettes in which the capsid protein and glycoproteins areseparated, used to derive stable split structural gene packaging celllines.

FIG. 21C is a western blot analysis demonstrating induction ofalphavirus capsid protein synthesis by several clonal cell linesfollowing transfection and subsequent synthesis of alphavirusglycoproteins or βgal from an alphavirus expression vector (ELVS-1.5PE[1.5 PE], or ELVS-βgal [βgal]).

FIG. 21D is a western blot analysis demonstrating induction ofalphavirus capsid protein synthesis by several clonal cell linesfollowing transfection and subsequent expression with an alphavirusexpression vector (ELVS-βgal).

FIG. 22 is a schematic illustration of the region of structural proteinexpression cassettes comprising a wild-type or deletion mutant RossRiver virus capsid protein gene.

FIG. 23 is a schematic illustration of vector inducible structuralprotein expression cassettes containing a wild-type or deletion mutantRoss River virus capsid protein gene.

FIG. 24 is a schematic illustration of vector packaging by “split”structural protein gene expression cassettes which contain a Ross Rivervirus capsid protein gene sequence upstream of the Sindbis virusglycoprotein genes.

FIG. 25 is a schematic illustration of vector packaging by “split”structural protein gene expression cassettes which contain a Ross Rivervirus capsid protein gene sequence upstream of the Sindbis virusglycoprotein genes on one cassette, and the Sindbis virus capsid proteingene in a separate cassette.

FIG. 26A is a table showing the results of vector particle packagingusing the above “split” structural protein gene expression cassettes.

FIG. 26B is two graphs which depict the packaging activity of 25 clonalcell lines from drug-resistant cell pools derived by stable transfectionwith the split structural protein gene expression cassettes illustratedin FIG. 21B, relative to a genomic structural protein gene PCL (987dlneo).

FIG. 26C is a western blot analysis demonstrating induction ofstructural protein expression by three split structural gene alphaviruspackaging cell lines following transfection and subsequent expressionwith an alphavirus vector (ELVS-βgal), but not a conventional plasmidDNA expression vector (pCI-βgal).

FIG. 26D is a graph depicting the amplification and production of β-galprotein over time in several split structural gene alphavirus packagingcell lines (Clone 9TD, Clone 2TD, Clone 24TD, Clone 20SS), relative to agenomic structural protein gene PCL (987 genomic PCL).

FIG. 27 is a schematic illustration of the use of alphavirus packagingcell lines for the amplification of packaged vector particlepreparations and the large scale production of recombinant protein.

FIG. 28 is a graph depicting the amplification and production of β-galprotein over time using alphavirus packaging cell lines.

FIG. 29 is a schematic illustration of the use of a tetracyclineregulated promoter system to control expression of alphavirus vector RNAfrom cDNA in vivo.

FIG. 30 is a schematic illustration of the use of a linkedtranscriptional repressor and a transcriptional inducer/activatorregulated promoter system to control expression of alphavirus vector RNAfrom cDNA in vivo.

FIGS. 31A & B are autoradiographs of [³H]uridine-labeled RNAselectrophoresed on denaturing glyoxal gels that were isolated from BHKcell electroporated with SINrep/LacZ replicon and DH RNAs from variousRRV capsid containing DH constructs, and from the vector particlespresent in the culture fluids at 18 hours post electroporation.

FIGS. 32A & B are protein gel autoradiographs depicting ³⁵Smethionine-labelled proteins from BHK cells electroporated withSINrep/LacZ replicon and DH RNAs from various RRV capsid containing DHconstructs, and from the vector particles present in the culture fluidsat 18 hours post electroporation.

FIGS. 33A-D are Kyte-Doolittle hydrophobicity plots of various RossRiver virus (RRV) capsid proteins, expressed from the wild-type gene (A)and three deletion mutants CΔ1rrv, CΔ2rrv, and CΔ3rrv (B-D,respectively).

FIG. 34 is a schematic that illustrates the amino-terminus RRV capsidproteins expressed from the wild-type gene (SEQ. ID NO. 114), and threedeletion mutants CΔ1rrv (SEQ. ID NO. 115), CΔ2rrv (SEQ. ID NO. 116), andCΔ3rrv (SEQ. ID NO. 117). The lysine residues deleted in the RRV capsidgene mutants are indicated.

FIG. 35 is a graph that illustrates the relative levels of[³⁵S]methionine and [³H]uridine incorporated into virus particles in BHKcells infected at high MOI with Toto1101 wild-type virus.

FIG. 36 is a graph that illustrates the relative levels of [³⁵S]methionine and [³H]uridine incorporated into virus particles in BHKcells electroporated with SINrep/lacZ and DH-BB (5′ tRNA) Crrv DH RNAs.

FIG. 37 is a graph that illustrates the relative levels of[³⁵S]methionine and [³H]uridine incorporated into virus particles in BHKcells electroporated with SINrep/lacZ and RRV capsid deletion mutantDH-BB (5′ tRNA) CΔ3rrv DH RNAs.

FIG. 38 is a graph which compiles the results shown in FIGS. 35-37,depicting the relative levels of [³⁵S]methionine and [³H]uridineincorporated into virus particles in BHK cells electroporated withSINrep/lacZ and DH RNAs, or infected with Toto1101 wild-type virus.

FIG. 39 is a graph which illustrates luciferase vector packaging by BHKcells stably transformed with pBGSVCMVdlhyg.

DEFINITION OF TERMS

The following terms are used throughout the specification. Unlessotherwise indicated, these terms are defined as follows:

“Genomic RNA” refers to RNA which contains all of the geneticinformation required to direct its own amplification or self-replicationin vivo, within a target cell. To direct its own replication, the RNAmolecule may: 1) encode one or more polymerase, replicase, or otherproteins which may interact with viral or host cell-derived proteins,nucleic acids or ribonucleoproteins to catalyze the RNA amplificationprocess; and 2) contain cis RNA sequences required for replication,which may be bound during the process of replication by its self-encodedproteins, or non-self-encoded cell-derived proteins, nucleic acids orribonucleoproteins, or complexes between any of these components. Analphavirus-derived genomic RNA molecule should contain the followingordered elements: 5′ viral or defective-interfering RNA sequence(s)required in cis for replication, sequences which, when expressed, codefor biologically active alphavirus nonstructural proteins (e.g., nsP1,nsP2, nsP3, nsP4), 3′ viral sequences required in cis for replication,and a polyadenylate tract. The alphavirus-derived genomic RNA vectorreplicon also may contain a viral subgenomic “junction region” promoterwhich may, in certain embodiments, be modified in order to prevent,increase, or reduce viral transcription of the subgenomic fragment, andsequences which, when expressed, code for biologically active alphavirusstructural proteins (e.g., C, E3, E2, 6K, E1). Generally, the termgenomic RNA refers to a molecule of positive polarity, or “message”sense, and the genomic RNA may be of length different from that of anyknown, naturally-occurring alphavirus. In preferred embodiments, thegenomic RNA does not contain the sequences which encode any alphaviralstructural protein(s); rather those sequences are substituted withheterologous sequences. In those instances where the genomic RNA is tobe packaged into a recombinant alphavirus particle, it must contain oneor more sequences which serve to initiate interactions with alphavirusstructural proteins that lead to particle formation, and preferably isof a length which is packaged efficiently by the packaging system beingemployed.

“Subgenomic RNA”, or “26S” RNA, refers to a RNA molecule of a length orsize which is smaller than the genomic RNA from which it was derived.The subgenomic RNA should be transcribed from an internal promoter whosesequences reside within the genomic RNA or its complement. Transcriptionof the subgenomic RNA may be mediated by viral-encoded polymerase(s),host cell-encoded polymerase(s), transcription factor(s),ribonucleoprotein(s), or a combination thereof. In preferredembodiments, the subgenomic RNA is produced from a vector according tothe invention, and encodes or expresses the gene(s) or sequencers) ofinterest. The subgenomic RNA need not necessarily have a sedimentationcoefficient of 26.

“Alphavirus vector construct” refers to an assembly which is capable ofdirecting the expression of a sequence(s) or gene(s) of interest. Suchvector constructs are comprised of a 5′ sequence which is capable ofinitiating transcription of an alphavirus RNA (also referred to as 5′CSE, in background), as well as sequences which, when expressed, codefor biologically active alphavirus nonstructural proteins (e.g., nsP1,nsP2, nsP3, nsP4), and an alphavirus RNA polymerase recognition sequence(also referred to as 3′ CSE, in background). In addition, the vectorconstruct should include a viral subgenomic “junction region” promoterwhich may, in certain embodiments, be modified in order to prevent,increase, or reduce viral transcription of the subgenomic fragment, andalso a polyadenylate tract. The vector also may include sequences fromone or more structural protein genes or portions thereof, extraneousnucleic acid molecule(s) which are of a size sufficient to allowproduction of viable virus, a 5′ promoter which is capable of initiatingthe synthesis of viral RNA in vitro from cDNA, a heterologous sequenceto be expressed, as well as one or more restriction sites for insertionof heterologous sequences.

“Alphavirus RNA vector replicon”, “RNA vector replicon” and “replicon”refers to a RNA molecule which is capable of directing its ownamplification or self-replication in vivo, within a target cell. Todirect its own amplification, the RNA molecule may: 1) encode one ormore polymerase, replicase, or other proteins which may interact withviral or host cell-derived proteins, nucleic acids or ribonucleoproteinsto catalyze RNA amplification; and 2) contain cis RNA sequences requiredfor replication which may be bound by its self-encoded proteins, ornon-self-encoded cell-derived proteins, nucleic acids orribonucleoproteins, or complexes between any of these components. Incertain embodiments, the amplification also may occur in vitro. Analphavirus-derived RNA vector replicon molecule should contain thefollowing ordered elements: 5′ viral sequences required in cis forreplication (also referred to as 5′ CSE, in background), sequenceswhich, when expressed, code for biologically active alphavirusnonstructural proteins (e.g., nsP1, nsP2, nsP3, nsP4), 3′ viralsequences required in cis for replication (also referred to as 3′ CSE,in background), and a polyadenylate tract. The alphavirus-derived RNAvector replicon also may contain a viral subgenomic “junction region”promoter which may, in certain embodiments, be modified in order toprevent, increase, or reduce viral transcription of the subgenomicfragment, sequences from one or more structural protein genes orportions thereof, extraneous nucleic acid molecule(s) which are of asize sufficient to allow production of viable virus, as well asheterologous sequence(s) to be expressed. The source of RLNA vectorreplicons in a cell may be from infection with a virus or recombinantalphavirus particle, or transfection of plasmid DNA or in vitrotranscribed RNA.

“Recombinant Alphavirus Particle” refers to a virion unit containing analphavirus RNA vector replicon. Generally, the recombinant alphavirusparticle comprises one or more alphavirus structural proteins, a lipidenvelope and an RNA vector replicon. Preferably, the recombinantalphavirus particle contains a nucleocapsid structure that is containedwithin a host cell-derived lipid bilayer, such as a plasma membrane, inwhich alphaviral-encoded envelope glycoproteins are embedded. Theparticle may also contain other components (e.g., targeting elementssuch as biotin, other viral structural proteins, or other receptorbinding ligands) which direct the tropism of the particle from which thealphavirus was derived, or other RNA molecules.

“Structural protein expression cassette” refers to a nucleic acidmolecule which is capable of directing the synthesis of one or morealphavirus structural proteins. The expression cassette should include a5′ promoter which is capable of initiating in vivo the synthesis of RNAfrom cDNA, as well as sequences which, when expressed. code for one ormore biologically active alphavirus structural proteins (e.g., C, E3,E2, 6K, E1), and a 3′ sequence which controls transcription termination.The expression cassette also may include a 5′ sequence which is capableof initiating transcription of an alphavirus RNA (also referred to as 5′CSE, in background), a viral subgenomic “junction region” promoter, andan alphavirus RNA polymerase recognition sequence (also referred to as3′ CSE, in background). In certain embodiments, the expression cassettealso may include splice recognition sequences, a catalytic ribozymeprocessing sequence, a sequence encoding a selectable marker, a nuclearexport signal, as well as a polyadenylation sequence. In addition,expression of the alphavirus structural protein(s) may, in certainembodiments, be regulated by the use of an inducible promoter.

“Stable Transformation” refers to the introduction of a nucleic acidmolecule into a living cell, and long-term or permanent maintenance ofthat nucleic acid molecule in progeny cells through successive cycles ofcell division. The nucleic acid molecule may be maintained in anycellular compartment, including, but not limited to, the nucleus,mitochondria, or cytoplasm. In preferred embodiments, the nucleic acidmolecule is maintained in the nucleus. Maintenance may beintrachromosomal (integrated) or extrachromosomal, as an episomal event.“Alphavirus packaging cell line” refers to a cell which contains analphavirus structural protein expression cassette and which producesrecombinant alphavirus particles after introduction of an alphavirusvector construct, RNA vector replicon, eukaryotic layered vectorinitiation system, or recombinant alphavirus particle. The parental cellmay be of mammalian or non-mammalian origin. Within preferredembodiments, the packaging cell line is stably transformed with thestructural protein expression cassette.

“Aliphavirus producer cell line” refers to a cell line which is capableof producing recombinant alphavirus particles, comprising an alphaviruspackaging cell line which also contains an alphavirus vector construct,RNA vector replicon, eukaryotic layered vector initiation system, orrecombinant alphavirus particle. Preferably, the alphavirus vectorconstruct is eukaryotic layered vector initiation system, and theproducer cell line is stably transformed with the vector construct. Inpreferred embodiments, transcription of the alphavirus vector constructand subsequent production of recombinant alphavirus particles occursonly in response to one or more factors, or the differentiation state ofthe alphavirus producer cell line.

“Defective helper construct” refers to an assembly which is capable ofRNA amplification or replication, and expression of one or morealphavirus structural proteins in response to biologically activealphavirus nonstructural proteins supplied in trans. The defectivehelper construct should contain the following ordered elements: 5′ viralor defective-interfering RNA sequences required in cis for replication,a viral subgenomic junction region promoter, sequences which, whenexpressed, code for one or more biologically active alphavirusstructural proteins (e.g., C, E3, E2, 6K, E1), 3′ viral sequencesrequired in cis for replication, and a polyadenylate tract. Thedefective helper construct also may contain a 5′ promoter which iscapable of initiating the synthesis of viral RNA from cDNA, a 3′sequence which controls transcription termination, splice recognitionsequences, a catalytic ribozyme processing sequence a sequence encodinga selectable marker, and a nuclear export signal.

“Eukaryotic Layered Vector Initiation System” refers to an assemblywhich is capable of directing the expression of a sequence(s) or gene(s)of interest. The eukaryotic layered vector initiation system shouldcontain a 5′ promoter which is capable of initiating in vivo (i.e.within a cell) the synthesis of RNA from cDNA, and a nucleic acid vectorsequence which is capable of directing its own replication in aeukaryotic cell and also expressing a heterologous sequence. The nucleicacid sequence which is capable of directing its own amplification may beof viral or non-viral origin. In certain embodiments, the nucleic acidvector sequence is an alphavirus-derived sequence and is comprised of a5′ sequence which is capable of initiating transcription of analphavirus RNA (also referred to as 5′ CSE, in background), as well assequences which, when expressed, code for biologically active alphavirusnonstructural proteins (e.g., nsP1, nsP2, nsP3, nsP4), and an alphavirusRNA polymerase recognition sequence (also referred to as 3′ CSE, inbackground). In addition, the vector sequence may include a viralsubgenomic “junction region” promoter which may, in certain embodiments,be modified in order to prevent, increase, or reduce viral transcriptionof the subgenomic fragment, sequences from one or more structuralprotein genes or portions thereof, extraneous nucleic acid molecule(s)which are of a size sufficient to allow optimal amplification, aheterologous sequence to be expressed, one or more restriction sites forinsertion of heterologous sequences, as well as a polyadenylationsequence. The eukaryotic layered vector initiation system may alsocontain splice recognition sequences, a catalytic ribozyme processingsequence, a nuclear export signal, and a transcription terminationsequence. In certain embodiments, in vivo synthesis of the vectornucleic acid sequence from cDNA may be regulated by the use of aninducible promoter.

“Alphavirus cDNA vector construct” refers to an assembly which iscapable of directing the expression of a sequence(s) or gene(s) ofinterest. The vector construct is comprised of a 5′ sequence which iscapable of initiating transcription of an alphavirus RNA (also referredto as 5′ CSE), as well as sequences which, when expressed, code forbiologically active alphavirus nonstructural proteins (e.g., nsP1, nsP2,nsP3, nsP4), and an alphavirus RNA polymerase recognition sequence (alsoreferred to as 3′ CSE, in background). In addition, the vector constructshould include a 5′ promoter which is capable of initiating in vivo thesynthesis of viral RNA from cDNA, and a 3′ sequence which controlstranscription termination. Within certain embodiments, the vectorconstruct may further comprise a viral subgenomic “junction region”promoter which may, in certain embodiments, be modified in order toprevent, increase, or reduce viral transcription of the subgenomicfragment The vector also may include sequences from one or morestructural protein genes or portions thereof, extraneous nucleic acidmolecule(s) which are of a size sufficient to allow production of viablevirus, a heterologous sequence to be expressed, one or more restrictionsites for insertion of heterologous sequences, splice recognitionsequences, a catalytic ribozyme processing sequence, a nuclear exportsignal, as well as a polyadenylation sequence. In certain embodiments,in vivo synthesis of viral RNA from cDNA may be regulated by the use ofan inducible promoter.

“Gene delivery vehicle” refers to a construct which can be utilized todeliver a gene or sequence of interest. Representative examples includealphavirus RNA vector replicons, alphavirus vector constructs,eukaryotic layered vector initiation systems and recombinant alphavirusparticles.

Numerous aspects and advantages of the invention will be apparent tothose skilled in the art upon consideration of the following detaileddescription which provides illumination of the practice of theinvention.

DETAILED DESCRIPTION OF THE INVENTION

As noted above, the present invention provides novel gene deliveryvehicles including for example, RNA vector replicons, alphavirus vectorconstructs, eukaryotic layered vector initiation systems and recombinantalphavirus particles. Briefly, introduction of plasmid DNA-, in vitrotranscribed RNA-, or particle-based vectors of the present inventioninto a cell, results in levels of heterologous gene expression that areequivalent, or higher, as compared to expression levels of wild-typederived alphaviral vectors. Unexpectedly however, the level ofvector-specific RNA synthesized is at least about 5 to 10-fold lower incultured cells which contain a gene delivery vehicle of the presentinvention as compared to wild-type derived vectors. Furthermore, suchgene delivery vehicles exhibit reduced, delayed, or no inhibition ofhost cell-directed macromolecular synthesis following introduction intoa host cell, as compared to wild-type derived vectors.

As discussed in more detail below, the present invention provides: (A)sources of wild-type alphaviruses suitable for constructing the genedelivery vehicles of the present invention; (B) methods for selectingalphaviruses with a desired phenotype; (C) construction of alphavirusvector constructs and alphavirus RNA vector replicons; (D) constructionof Eukaryotic Layered Vector Initiation Systems; (E) construction ofrecombinant alphavirus particles; (F) heterologous sequences which maybe expressed by the gene delivery vehicles of the present invention; (G)construction of alphavirus packaging or producer cell lines; (H)pharmaceutical compositions; and (I) methods for utilizingalphavirus-based vectors.

A. Sources of Wild-Type Alphavirus

As noted above, the present invention provides a wide variety ofalphavirus-based vectors (e.g., RNA vector replicons, alphavirus vectorconstructs, eukaryotic layered vector initiation systems and recombinantalphavirus particles), as well as methods for utilizing such vectorconstructs and particles. Briefly, sequences encoding wild-typealphaviruses suitable for use in preparing the above-described vectorscan be readily obtained given the disclosure provided herein fromnaturally-occurring sources, or from depositories (e.g., the AmericanType Culture Collection, Rockville, Md.). In addition, wild-typealphaviruses may be utilized for comparing the level of host-celldirected macromolecular synthesis in cells infected with the wild-typealphavirus, with the level of host-cell directed macromolecularsynthesis in cells containing the gene delivery vehicles of the presentinvention.

Representative examples of suitable alphaviruses include Aura virus(ATCC VR-368), Bebaru virus (ATCC VR-600, ATCC VR-1240), Cabassou virus(ATCC VR-922), Chikungunya virus (ATCC VR-64, ATCC VR-1241), Easternequine encephalomyelitis virus (ATCC VR-65. ATCC VR-1242), Fort Morganvirus (ATCC VR-924), Getah virus (ATCC VR-369, ATCC VR-1243), Kyzylagachvirus (ATCC VR-927), Mayaro virus (ATCC VR-66, ATCC VR-1277), Middleburgvirus (ATCC VR-370), Mucambo virus (ATCC VR-580, ATCC VR-1244), Ndumuvirus (ATCC VR-371), Pixuna virus (ATCC VR-372, ATCC VR-1245), RossRiver virus (ATCC VR-373, ATCC VR-1246), Semliki Forest virus (ATCCVR-67, ATCC VR-1247), Sindbis virus (ATCC VR68, ATCC VR-1248; see alsoCMCC #4640, described below), Tonate virus (ATCC VR-925), Triniti virus(ATCC VR-469), Una virus (ATCC VR-374), Venezuelan equineencephalomyelitis virus (ATCC VR-69, ATCC VR-923, ATCC VR-1250 ATCCVR-1249, ATCC VR-532), Western equine encephalomyelitis virus (ATCCVR-70, ATCC VR-1251, ATCC VR-622, ATCC VR-1252), Whataroa virus (ATCCVR-926), and Y-62-33 virus (ATCC VR-375).

For purposes of comparing levels of cellular macromolecular synthesis,the following plasmids may also be utilized as a standard source ofwild-type alphavirus stocks. These plasmids include: for Semliki ForestVirus, pSP6-SFV4 (Liljestrom et al., J Virol. 65:4107-4113,1991); forVenezuelan equine encephalitis virus, pV2000 (Davis et al., Vir.183:20-31,1991); for Ross River virus, pRR64 (Kuhn et al., Vir.182:430-441,1991). Briefly, for these plasmids, virus can be obtainedfrom BHK cells transfected with in vitro transcribed genomic RNA fromthe plasmids. For Sindbis virus, infectious virus may be isolateddirectly from BHK cells transfected with pVGELVIS (ATCC No. 75891)plasmid DNA, or alternatively, obtained as a wild-type virus stock (seedeposit information provided below regarding ATCC No. VR-2526).

B. Selection of Alphaviruses With a Desired Phenotype

The duration of in vivo heterologous gene expression fromalphavirus-based vectors is affected by several mechanisms, includinginhibition of host cell-directed macromolecular synthesis. However,prior to the present invention, there had been no obvious method toselect for or identify coding or non-coding vector viral-specificsequence changes that result in a non-cytopathic phenotype. Therefore,within one aspect of the present invention methods are provided forisolating and/or constructing alphavirus-derived gene delivery vehicleswith reduced or no inhibition of host cell directed macromolecularsynthesis.

1. Biological Selection of Virus Variants

a. Selection from Virus Stocks Containing DI Particles

One approach for isolating non-cytopathic alphavirus variants exploitsthe presence of defective interfering (DI) particles in wild-type viruspreparations. Briefly, although certain RNA viruses, for examplerhabdoviruses (e.g., vesicular stomatitis virus) and alphaviruses (e.g.,Sindbis virus and Semliki Forest virus), are highly cytopathic, they cannevertheless establish long-term persistent infection in cultured cellsin the presence of DI particles. DI particles, by definition, arederived from wild-type virus and contain one or more mutations (e.g.,deletions, rearrangements, nucleotide substitutions, etc.) from thewild-type genome which prevent autonomous replication by the DI. Ingeneral, the genome of DI particles is smaller and of a lower complexitycompared to wild-type virus, and is deleted of protein-encoding regionswhile maintaining regions required in cis for replication. Such cissequences often are duplicated and/or rearranged. In the case of certainalphaviruses (e.g., Sindbis virus), the sequence and organization of DIRNA genomes have been analyzed and found to contain a minimum of 50 ntfrom the extreme 3′ -end of the wild-type virus genome, and at their 5′-ends, either a wild-type sequence or a cellular tRNA (e.g., TRNA^(asp))sequence, in addition to the viral sequence. In all cases, thepropagation and maintenance of the mutated DI genomes requires theco-existence of parental helper virus in the infected cell. However, asa result of their genetic structure, DI genome replication is vastlysuperior and comparatively abundant to its wild-type counterpart. Thischaracteristic results in interference of wild-type genome replication,the absence or low level production of infectious virus, and theestablishment of long-term persistent infection of cells.

Therefore, as described below in Examples 1 and 2, the ability toestablish long-term persistent infection in permissive cells (e.g.,mammalian cells, including cells of human origin) by infecting with amixed alphavirus stock containing a population of DI particles providesa mechanism to isolate, over time, fully intact virus variants that areable to establish persistent infection even in the absence of DIparticles. Such infectious virus variants can be isolated from long-termpersistently infected cultures by multiple rounds of plaque purificationand have been found to initiate productive, persistent, andnon-cytopathic infection in the host cells. Furthermore, the level ofvariant virus produced from such a productive, persistent, andnon-cytopathic infection is indistinguishable from wild-type virusinfection. This observation is in distinct contrast to the previousrequirement for establishment of persistent infections with virus stockscontaining a mixture of DI particles.

b. Selection from Virus Stocks Not Containing DI Particles

In addition to selection from virus stocks that containdefective-interfering particles, virus variants suitable for use withinthe present invention may be obtained from purified virus stocks(without DI particles) which are either subjected to random mutagenesisprior to infection of susceptible cultured cells or allowed to generatenon-specific mutations during RNA replication with the cultured cells.Briefly, the initial virus stock may be obtained as a natural isolate orbiological variant derived therefrom, or may be generated bytransfecting cultured cells with an infectious nucleic acid moleculecomprising a genomic cDNA clone or in vitro transcribed RNA. If desired,the virus stock may then subjected to physical or chemical mutagenesis(although preferred, such mutagenesis is not required). In the case ofchemical mutagenesis, preferred embodiments utilize a readily availablemutagenic agent, for example nitrous acid, 5-azacytidine,N-methyl-N′-nitro-N-nitrosoguanidine, or ethylmethane sulfonate (Sigma,St. Louis, Mo.), prior to virus infection. Following random mutagenesis,specific selection procedures are applied to isolate virus variantspossessing the desired phenotype, as described in more detail below inExample 2.

2. Genetic Selection of Virus Variants

In a related approach, mutations may be obtained not using a virusstock, but rather, using cloned genomic cDNA of the virus that can beused subsequently to transcribe infectious viral RNA in vitro (forexample, Sindbis virus (Rice et al., J Virol. 61:3809-3819, 1987;Dubensky et al., J. Virol. 70:508-519, 1996, SFV (Liljeström et al., J.Virol. 65:4107-4113., 1991, VEE (Davis et al., Virology 183:20-31,1991), Ross River virus (Kuhn et al., Virology 182:430-441 1991),poliovirus (Van Der Werf et al., Proc. Natl. Acad. Sci. USA83:2330-2334, 1986)) or in vivo (Sindbis virus (Dubensky et al., ibid.),poliovirus (Racaniello and Baltimore, Science 214:916-919, 1981)).Briefly, the infectious nucleic acid is introduced into susceptiblecultured cells (e.g., mammalian cells, including cells of human origin)either directly or following mutagenesis performed using one of theabove-referenced methods. Alternatively, vector nucleic acid may bepackaged into particles initially, and the particles used to delivervector into the target cell population for selection. Subsequently,specific selection procedures to isolate virus variants possessing thedesired phenotype are applied, and are described below.

In certain embodiments, random mutagenesis may be performed initially bypropagation of the plasmid containing viral cDNA in the XL1-Red strainof E. coli (Stratagene, San Diego, Calif.), which is deficient in threeof the primary DNA repair pathways, resulting from mutS, mutD, and mutTmutations. However, other mutagenesis procedures including, but notlimited to, linker-scanning mutagenesis (Haltiner et al., Nucleic AcidsRes. 13:1015, 1985; Barany, Proc. Natl. Acad. Sci. USA 82:4202, 1985),random oligonucleotide-directed mutagenesis (Kunkel et al., MethodsEnzymol. 155:166, 1987; Zoller and Smith, Methods Enzymol. 154:329,1987; Hill et al., Methods Enzymol. 155:558, 1987; Hermes et al., Gene84:143, 1989) and PCR mutagenesis (Herlitze and Koenen, Gene 91:143,1990), can be readily substituted utilizing published protocols. Theresulting mixed population of mutated CDNA clones is introduced intosusceptible cultured cells directly, or after transcription in vitro.Enrichment for transfected cells which contain mutated virus of thedesired phenotype is accomplished based on increased survival time overwild-type virus infected cells, as described below.

3. Genetic Selection of Variants Using Virus-Derived Vectors

In another approach, mutations may be generated in any region avirus-derived expression vector, including the regulatory, untranslatedregions, or protein-encoding gene regions. For example, within oneaspect of the invention methods are provided for selecting viralvariants with reduced or no inhibition of host-cell directedmacromolecular synthesis, comprising the steps of: (a) introducing intoa cell a eukaryotic layered vector initiation system, RNA vectorreplicon, or recombinant alphavirus particle which directs theexpression of a immunogenic cell surface protein (suitable for detectionof vector containing cells), or alternatively, a selectable marker(either a drug or non-drug marker wherein non-vector containing cellsare killed upon addition of, for example, a drug such as neomycin,hygromycin, phleomycin, gpt, puromycin, or histidinol); (b) incubationor culturing the cells under conditions and for a time sufficient toselect vector containing cells which exhibit the desired phenotype;followed by (c) isolating cells which contain the vector of the desiredphenotype and (d) analysis of the vector for the causal mutation.

As noted above, the viral vectors of the present invention may bederived from a wide variety of viruses (e.g., Sindbis virus (Xiong etal., Science 243:1188-1191, 1989; Hahn et al., Proc. Natl. Acad. Sci.USA 89:2679-2683, 1992; Schlesinger, Trends Biotechnol. 11:18-22,1993;Dubensky et al., ibid), Semliki Forest virus (Liljestrom andGaroff, Bio/Technology 9:1356-1361, 1991), Venezuelan equineencephalitis virus (Davis et al., J. Cell. Biochem. Suppl. 19A:310,1995), poliovirus (Choi et al., J. Virol 65:2875-2883, 1991; Ansardi etal., Cancer Res. 54:6359-6364, 1994; and Andino et al., Science265:1448-1451, 1994). Representative examples of the above-describedmethods are discussed-in more detail below within Example 2.

4. Use of Viral Variants

As discussed in more detail herein, viral variants which have beenselected or generated utilizing the methods provided herein may beutilized to construct a wide variety of recombinant gene deliveryvehicles which exhibit the desired phenotype. Within certainembodiments, the gene delivery vehicle contains a mutation within theLeu-Xaa-Pro-Gly-Gly (“LXPGG”) motif of the nsP2 gene. Briefly, foralphaviruses wherein published sequence of the nsP2 gene is available, ahighly conserved amino acid motif -Leu-Xaa-Pro-Gly-Gly- (“LXPGG”) isobserved. As predicted by standard protein modeling algorithms (Chou andFasman, Adv. Enzym. 47:45-148, 1978), the residues of this motifpossibly comprise a β turn in the structure. Proline 726 of nsP2 inSindbis virus is the central residue of this motif. The correspondingmotif in other alphaviruses is illustrated in the table below.

Alphavirus Strain* Pro-Gly-Gly Region nsP2 a.a.'s (P-G-G) 1. Sindbisvirus Leu-Asn-Pro-Gly-Gly-Thr a.a. = 726-728 2. S.A.AR86 virusLeu-Asn-Pro-Gly-Gly-Thr a.a. = 726-728 3. Ockelbo virusLeu-Asn-Pro-Gly-Gly-Thr a.a. = 726-728 4. Aura virusLeu-Lys-Pro-Gly-Gly-Thr a.a. = 725-727 5. Semliki Forest virusLeu-Lys-Pro-Gly-Gly-Ile a.a. = 718-720 6. VEE virusLeu-Asn-Pro-Gly-Gly-Thr a.a. = 713-715 7. Ross River virusLeu-Xaa-Pro-Gly-Gly-Ser a.a. = 717-719 *Alphavirus strains withpublished nsP2 sequences: (1) Strauss et al., Virology 133:92-110, 1984;(2) Simpson et al., Virology 222:464-469 1996; (3) Shirako et al.,Virology 182:753-764, 1991; (4) Rumenapf et al., Virology 208:621-633,1995; (5) Takkinen, Nucleic Acids Res. 14:5667-5682, 1986; (6) Kinney etal., Virology 170:19-30, 1989; and (7) Faragher et al, Virology163:509-526, 1988.

Hence, within various embodiments of the present invention, genedelivery vehicles are provided wherein the gene delivery vehiclecontains an nsP2 gene with a mutation in the LXPGG motiff. Within oneembodiment, the Leu codon is mutated to another amino acid selected fromthe group consisting of Ala, Arg, Asn, Asp, Asx, Cys, Gln, Glu, Glx,Gly, His, Ile, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, or anotherrare or non-protein amino acid (see, e.g., Lehninger, Biochemistry,Worth Publishers. Inc., N.Y. N.Y., 1975). Within another embodiment, thePro codon is mutated to another amino acid selected from the groupconsisting of Ala, Arg, Asn; Asp, Asx, Cys, Gln, Glu, Glx, Gly, His,Ile, Leu, Lys, Met, Phe, Ser, Thr, Trp, Tyr, Val, or another rare ornon-protein amino acid. Within other embodiments, either or both of theGly codons may be mutated to another amino acid selected from the groupconsisting of Ala, Arg, Asn, Asp, Asx, Cys, Gln, Glu, Glx, His, Ile,Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, Val, or another rare ornon-protein amino acid. Within yet other embodiments, the Xaa aminoacid, or amino acids between 1 and 3 residues upstream or downstream ofthe LXPGG motiff may be mutated from the wild-type amino acid in orderto effect the phenotype of the resultant gene delivery vehicle. Withincertain embodiments of the invention, the LXPGG motiff may be mutated tocontain more than one codon alteration, or alternatively, one or morecodon insertions or deletion.

C. Alphavirus Vector Constructs and Alphavirus RNA Vector Replicons

As noted above, the present invention provides both DNA and RNAconstructs which are derived from alphaviruses. Briefly, within oneaspect of the present invention alphavirus vector constructs areprovided, comprising a 5′ promoter which initiates synthesis of viralRNA in vitro from cDNA, a 5′ sequence which initiates transcription ofalphavirus RENAL a nucleic acid molecule which operably encodes all fouralphaviral nonstructural proteins including an isolated nucleic acidmolecule as described above, an alphavirus RNA polymerase recognitionsequence and a 3′ polyadenylate tract. Within other aspects, RNA vectorreplicons are provided. comprising a 5′ sequence which initiatestranscription of alphavirus RNA, a nucleic acid molecule which operablyencodes all four alphaviral nonstructural proteins, including theisolated nucleic acid molecules discussed above, an alphavirus RNApolymerase recognition sequence and a 3′ polyadenylate tract. Withinpreferred embodiments of the above, the above constructs furthercomprise a viral junction region. Each of these aspects are discussed inmore detail below.

1. 5′ Promoters which initiate synthesis of viral RNA

As noted above, within certain embodiments of the invention, alphavirusvector constructs are provided which contain 5′ promoters which (e.g.,DNA dependent RNA polymerase promoters) initiate synthesis of viral RNAfrom CDNA by a process of in vitro transcription. Within preferredembodiments such promoters include, for example, the bacteriophage T7,T3, and SP6 RNA polymerase promoters. Similarly, eukarytoic layeredvector initiation systems are provided (e.g., DNA dependent RNApolymerase promoters) which contain 5′ promoters which initiatesynthesis of viral RNA from cDNA in vivo (i.e., within a cell). Withincertain embodiments, such RNA polymerase promoters (for eitheralphavirus vector constructs or eukaryotic layered vector initiationsystems) may be derived from both prokaryotic and eukaryotic organisms,and include, for example, the bacterial β-galactosidase and trpEpromoters, and the eukaryotic viral simian virus 40 (SV40) (e.g., earlyor late), cytomegalovirus (CMV) (e.g., immediate early), Moloney murineleukemia virus (MOMLV) or Rous sarcoma virus (RSV) LTR, and herpessimplex virus (HSV) (thymidine kinase) promoters.

2. Sequences Which Initiate Transcription

As noted above, within preferred embodiments the alphavirus vectorconstructs and RNA vector replicons of the present invention contain a5′ sequence which is capable of initiating transcription of analphavirus RNA (also referred to as 5′-end CSE, or 5′ cis replicationsequence). Representative examples of such sequences include nucleotides1-60, and to a lesser extent nucleotides through bases 150-210, of thewild-type Sindbis virus, nucleotides 10-75 for tRNAsP (aspartic acid,Schlesinger et al., U.S. Pat. No. 5,091,309), and 5′ sequences fromother alphaviruses which initiate transcription. It is the complement ofthese sequences, which corresponds to the 3′ end of the of theminus-strand genomic copy, which are bound by the nsP replicase complex,and possibly additional host cell factors, from which transcription ofthe positive-strand genomic RNA is initiated.

3. Alphavirus Nonstructural Proteins

The alphavirus vector constructs and RNA vector replicons providedherein also require sequences encoding all four alphaviral nonstructuralprotein, including a sequence which provides the desired phenotypediscussed above. Briefly, a wide variety of sequences which encodealphavirus nonstructural proteins, in addition to those explicitlyprovided herein, may be utilized in the present invention, and aretherefore deemed to fall within the scope of the phrase “alphavirusnonstructural proteins.” For example, due to the degeneracy of thegenetic code, more than one codon may code for a given amino acid.Therefore, a wide variety of nucleic acid sequences which encodealphavirus nonstructural proteins may be generated. Furthermore, aminoacid substitutions, additions, or deletions at any of numerous positionsmay still provide functional or biologically active nonstructuralproteins. Within the context of the present invention, alphavirusnonstructural proteins are deemed to be biologically active if theypromote self-replication of the vector construct. i.e., replication ofviral nucleic acids and not necessarily the production of infectiousvirus, and may be readily determined by metabolic labeling or RNaseprotection assays performed over a time course. Methods for making suchderivatives are readily accomplished by one of ordinary skill in the artgiven the disclosure provided herein.

Alphaviruses express four nonstructural proteins, designated nsp1, nsp2,nsp3, and nsp4. Vectors of the present invention derived fromalphaviruses should contain sequences encoding the four nonstructuralproteins. In wild-type Sindbis virus, nonstructural proteins 1-3 areencoded by nucleotides 60 to 5747, while nsP4 is encoded by nucleotides5769 to 7598 (see FIG. 1). The nonstructural proteins are translatedfrom the genomic positive strand RNA as one of two large polyproteins,known as P123 or P1234, respectively, depending upon (i) whether thereis an opal termination codon between the coding regions of nsP3 and nsP4and (ii) if there is such an opal codon present, whether there istranslation termination of the nascent polypeptide at that point or readthrough and hence production of P1234. The opal termination codon ispresent at the nsP3/nsP4 junction of the alphaviruses SIN (strain AR339and the SIN-1 strain described herein), AURA, WEE, EEE, VEE, and RR, andthus the P123 and P1234 species are expressed in cells infected withthese viruses. In contrast, no termination codon is present at thensP3/nsP4 junction of the alphaviruses SIN (strain AR86, SF, and ONN),and thus only the P1234 species is expressed in cells infected withthese viruses. Both the polyprotein and processed monomeric forms of thenonstructural proteins function in the replication of the alphavirus RNAgenome. Experiments examining growth characteristics of alphavirusnonstructural protein cleavage mutants have indicated that thepolyproteins are involved in the synthesis of the genomic negativestranded RNA, while the individual monomeric proteins catalyze thesynthesis of the genomic and subgenomic positive stranded RNA species(Shirako and Strauss, J. Virol. 68:1874-1885, 1994). Translational readthrough generally occurs about 10%-20% of the time in cells infectedwith wild type Sindbis virus containing the opal termination codon atthe nsP3/nsP4 junction. Processing of P123 and P1234 is by a proteinaseactivity encoded by the one of the nonstructural proteins, and isdiscussed further below. The order of processing, whether in cis or intrans, depends on various factors, including the stage of infection. Forexample, Sindbis virus and SFV produce P123 and nsp4 early in infection,and P12 and P34 later in infection. Further processing then releases theindividual nonstructural proteins. Each nonstructural protein hasseveral functions, some of which are described below.

a. nsP1

Nonstructural protein 1 is required for the initiation of (orcontinuation of) minus-strand RNA synthesis. It also plays a role incapping the 5′ terminus of genomic and subgenomic alphavirus RNAs duringtranscription, as nsP1 possesses both methyltransferase (Mi and Stollar,Vir. 184:423-427, 1991) and guanyltransferase activity (Strauss andStrauss, Microbiol, Rev. 58(3):491-562, 1994). NsP1 also modulates theproteinase activity of nsP2, as polyproteins containing nsP1inefficiently cleave between nsP2 and nsP3 (de Groot et al., EMBO J.9:2631-2638, 1990).

b. nsP2

Nonstructural protein 2 is a multifunctional protein, involved in thereplication of the viral RNA and processing of the nonstructuralpolyprotein. The N-terminal domain of the protein (spanning about thefirst 460 amino acids) is believed to be a helicase which is active induplex unwinding during RNA replication and transcription. Synthesis of26S subgenomic mRNA, which, in vectors according to the presentinvention, encodes the gene(s) of interest, requires functional nsP2.The C-terminal domain of nsP2, between amino acid residues 460-807 ofSindbis virus, proteolytically cleaves in trans and in cis thenonstructural polyprotein between the nsP1/nsP2, nsP2/nsP3, andnsP3/nsP4 junctions. Alignment of the primary sequences of thealphavirus nsP2 C-termninal domains suggests that nsP2 is a papain-likeproteinase (Hardy and Strauss, J. Virol. 63:4653-4664, 1988).

Other observed characteristics of nsP2 have not, as yet, been assigned afunction directly related to the propagation of alphaviruses. Forexample, it has been shown that nsP2 is closely associated withribosomes in SFV-infected cells, and can be cross-linked to rRNA by UVirradiation (Ranki et al., FEBS Lett. 108:299-302, 1979). Further, 50%of nsP2 is localized in the nuclear matrix, particularly in the area ofthe nucleoli of SFV-infected BHK cells (Peränen et al., J. Virol.64:1888-1896, 1990). Localization of nsP2 to the nuclei presumablyproceeds by active transport, as it exceeds the size of small proteinsand metabolites (about 20-60 kD), which can enter the nucleus bydiffusion through nuclear core complexes (Paine et al., Nature254:109-114, 1975). Putative NLS sequences have been identified in thealphaviruses SFV, SIN, RR, ONN, OCK, and VEE (Rikkonen et al., Vir.189:462-473, 1992).

c. nsP3

Nonstructural protein nsP3 contains two distinct domains, although theirprecise roles in viral replication are not well understood. TheN-terminal domain ranges in length from 322 to 329 residues in differentalphaviruses and exhibits a minimum of 51% amino acid sequence identityamong any two alphaviruses. The C-terminal domain, however, is notconserved among known alphaviruses in length or in sequence, andmultiple changes are tolerated (Li et al., Virology, 179:416-427). Theprotein is found associated with replication complexes in a heavilyphosphorylated state. In alphaviruses whose genomes contain an opaltermination codon between the nsP3/nsP4 junction, two different proteinsare produced depending upon whether or not there is read through of theopal termination signal. Read through results in an nsP3 protein whichcontains 7 additional carboxy terminal amino acids after cleavage of thepolyprotein. It is clear that nsP3 is required in some capacity forviral RNA synthesis, as particular mutants of this protein are RNAnegative, and the P123 polyprotein is required for minus-strand RNAsynthesis.

d. nsP4

NsP4 is the virus-encoded RNA polymerase and contains the GDD motifcharacteristic of such enzymes (Kamer and Argos, Nucleic Acids Res.12:7269-7282, 1984). Thus, nsP4 is indispensable for alphavirus RNAreplication. The concentration of nsP4 is tightly regulated in infectedcells. In most alphaviruses, translation of nsP4 requires read throughof an opal codon between the nsP3 and nsP4 coding regions, resulting inlower intracellular levels as compared to other nonstructural proteins.Additionally, the bulk of nsP4 is metabolically unstable, throughdegradation by the N-end rule pathway (Gonda et al. J. Biol. Chem. 264:16700-16712, 1989). However, some nsP4 is stable, due to its associationwith replication complexes which conceal degradation signals. Thus,stabilization of the enzyme by altering the amino terminal residue mayprove useful in promoting more long term expression of proteins encodedby the vectors described herein. Stabilizing amino terminal residuesinclude methionine, alanine, and tyrosine.

4. Viral Junction Regions

The alphavirus viral junction region normally controls transcriptioninitiation of the subgenomic mRNA; thus, this element is also referredto as the subgenomic mRNA promoter. In the case of Sindbis virus, thenormal viral junction region typically begins at approximatelynucleotide number 7579 and continues through at least nucleotide number7612 (and possibly beyond). At a minimum, nucleotides 7579 to 7602(5′-ATC TCT ACG GTG GTC CTA AAT AGT-SEQ. ID NO. 1) are believednecessary for transcription of the subgenomic fragment. This region(nucleotides 7579 to 7602) is hereinafter referred to as the “minimaljunction region core.”

Within certain aspects of the invention, the viral junction region isinactivated in order to prevent synthesis of the subgenomic fragment. Asutilized within the context of the present invention, “inactivated”means that the species corresponding to subgenomic MRNA is not observedin autoradiograms from denaturing gels of electrophoresed RNA purifiedfrom cells containing these vectors and treated with 1 μg/mldactinomycin and labeled with [³H]-uridine, as described (Frolov andSchlesinger, J. Virol. 68:1721-1727, 1994).

Within one embodiment of the invention, gene delivery vehicles may beconstructed by the placement of signals promoting either ribosome readthrough or internal ribosome entry immediately downstream of thedisabled junction region promoter. In this vector configuration,synthesis of subgenomic message cannot occur; however, the heterologousproteins are expressed from genomic length mFNA by either ribosomal readthrough (scanning) or internal ribosome entry.

In certain applications of the gene delivery vehicles described herein,the expression of more than one heterologous gene is desired. Forexample, in order to treat metabolic disorders such as Gaucher'ssyndrome, multiple administrations of gene delivery vehicles orparticles may be required, since duration of the therapeutic palliativemay be limited. Therefore, within certain embodiments of the inventionit may be desirable to co-express in a target cell the Adenovirus 2 E3gene, along with a therapeutic palliative, such as theglucocerebrosidase gene. In wild-type virus, the structural protein (sP)polycistronic message is translated into a single polyprotein which isprocessed subsequently into individual proteins in part by the sP capsidproteinase. Thus, expression of multiple heterologous genes from apolycistronic message requires a mechanism different from the wild-typevirus, since the protease activity of the capsid sP, or the peptidesrecognized for cleavage, are not present in the replacement region ofthe alphavirus vectors. Therefore, within further embodiments of theinvention, functional elements which permit translation of multipleindependent heterologous sequences, including ribosomal read through,cap-independent translation, internal ribosome entry, or minimaljunction region core sequences, can be utilized.

5. Alphavirus RNA polymerase recognition sequence, and poly(A) tract

As noted above, alphavirus vector constructs or RNA vector replicons ofthe present invention also should include an alphavirus RNA polymeraserecognition sequence (also termed “alphavirus replicase recognitionsequence”, “3′ terminal CSE”, or “3′ cis replication sequence”).Briefly, the alphavirus RNA polymerase recognition sequence, which islocated at the 3′ end region of positive stranded genomic RNA, providesa recognition site at which the virus begins replication by synthesis ofthe negative strand. A wide variety of sequences may be utilized as analphavirus RNA polymerase recognition sequence. For example, within oneembodiment, Sindbis virus vector constructs in which the polymeraserecognition is truncated to the smallest region that can still functionas a recognition sequence (e.g., nucleotides 11, 684 to 11, 703) can beutilized. Within another embodiment of the invention, Sindbis virusvector constructs in which the entire nontranslated region downstreamfrom the E1 sP gene to the 3′ end of the viral genome including thepolymerase recognition site (e.g., nucleotides 11,382 to 11, 703), canbe utilized.

Within preferred embodiments of the invention, the alphavirus vectorconstruct or RNA vector replicon may additionally contain a poly(A)tract, which increases dramatically the observed level of heterologousgene expression in cells transfected with alphavirus-derived vectors(see e.g., Dubensky et al, supra). Briefly, the poly(A) tract may be ofany size which is sufficient to promote stability in the cytoplasm,thereby increasing the efficiency of initiating the viral life cycle.Within various embodiments of the invention, the poly(A) sequencecomprises at least 10 adenosine nucleotides, and most preferably, atleast 25 adenosine nucleotides. Within one embodiment, the poly(A)sequence is attached directly to Sindbis virus nucleotide 11, 703.

D. Eukaryotic Layered Vector Initiation Systems

Due to the size of a full-length genomic alphavirus cDNA clone, in vitrotranscription of full-length, capped RNA molecules is ratherinefficient. This results in a lowered transfection efficiency, in termsof infectious centers of virus (as measured by plaque formation),relative to the amount of in vitro transcribed RNA transfected. Suchinefficiency is also relevant to the in vitro transcription ofalphavirus expression vectors. Testing of candidate cDNA clones andother alphavirus cDNA expression vectors for their ability to initiatean infectious cycle or to direct the expression of a heterologoussequence can thus be greatly facilitated if a cDNA clone is transfectedinto susceptible cells as a DNA molecule, which then directs thesynthesis of viral RNA in vivo.

Therefore, within one aspect of the present invention DNA-based vectors(referred to as “Eukaryotic Layered Vector Initiation Systems”) areprovided that are capable of directing the synthesis- of viral RNA(genomic or vector) in vivo. Generally, eukaryotic layered vectorinitiation systems comprise a 5′ promoter that is capable of initiatingin vivo (i.e., within a cell) the 5′ synthesis of RNA from cDNA, aconstruct that is capable of directing its own replication in a cell,the construct also being capable of expressing a heterologous nucleicacid sequence, and a 3′ sequence that controls transcription termination(e.g., a polyadenylate tract). Such eukaryotic layered vector initiationsystems provide a two-stage or “layered” mechanism that controlsexpression of heterologous nucleotide sequences. Briefly, the firstlayer initiates transcription of the second layer and comprises apromoter that is capable of initiating in vilo the 5′ to 3′ synthesis ofRNA from cDNA (e.g., a 5′ eukaryotic promoter), and may further compriseother elements, including a 3′ transcription termination/polyadenylationsite, one or more splice sites, as well as other RNA nuclear exportelements, including, for example, the hepatitis B virusposttranscriptional regulatory element (PRE) (Huang et al., Mol. Cell.Biol. 13:7476, 1993; Huang et al., J Virol. 68:3193, 1994; Huang et al.,Mol. Cell. Biol., 15:3864-3869, 1995), the Mason-Pfizer monkey virusconstitutive transport element (CTE) (Bray et al., Proc. Natl. Acad.Sci. USA91:1256-1260, 1994), the HIV Rev responsive element (Malim etal., Nature 338:254-257, 1989; Cullen et al., Trends Biochem.Sci.16:346, 1991), and other similar elements, if desired.Representative promoters suitable for use within the present inventioninclude both eukaryotic (e.g., pol I, II, or III) and prokaryoticpromoters, and inducible or non-inducible (i.e., constitutive)promoters, such as, for example., Moloney murine leukemia viruspromoters, metallothionein promoters, the glucocorticoid promoter.Drosophila actin 5C distal promoter, SV40 promoter, heat shock protein65 promoter, heat shock protein 70 promoter, immunoglobulin promoters,mouse polyoma virus promoter (Py), Rous sarcoma Virus (RSV), herpessimplex virus (HSV) promoter, BK virus and JC virus promoters, mousemammary tumor virus (MMTV) promoter, alphavirus junction region, CMVpromoter, Adenovirus E1 or VA1RNA promoters, rRNA promoters, tRNAmethionine promoter, CaMV 35S promoter, nopaline synthetase promoter,tetracycline responsive promoter, and the lac promoter.

Within yet other embodiments of the invention, inducible promoters maybe utilized. For example, within one embodiment inducible promoters areprovided which initiate the synthesis of RNA from DNA, comprising a coreRNA polymerase promoter sequence, and an operably linked nucleic acidsequence that directs the DNA binding of a transcriptional activatorprotein, and an operably linked nucleic acid sequence that directs theDNA binding of a transcriptional repressor protein. Within a furtherembodiment, the nucleic acid sequence that directs the DNA binding of atranscriptional activator protein is a sequence that binds atetracycline repressor/VP16 transactivator fusion protein. Within yetanother embodiment, the nucleic acid sequence that directs the DNAbinding of a transcription repressor protein is a sequence that binds alactose repressor/Kruppel domain fusion protein.

The second layer comprises an autocatalytic vector construct which iscapable of expressing one or more heterologous nucleotide sequences andof directing its own replication in a cell, either autonomously or inresponse to one or more factors (e.g. is inducible). The second layermay be of viral or non-viral origin. Within one, embodiment of theinvention, the second layer construct may be an alphavirus vectorconstruct as described above.

A wide variety of vector systems may be utilized as the first layer ofthe eukaryotic layered vector initiation system, including for example,viral vector constructs developed from DNA viruses such as thoseclassified in the Poxviridae, including for example canary pox virus orvaccinia virus (e.g., Fisher-Hoch et al., PNAS 86:317-321, 1989; Flexneret al., Ann. N.Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine8:17-21, 1990; U.S. Pat. Nos. 4,603,112, 4,769,330 and 5, 017, 487; WO89/01973); Papoviridae such as BKV, JCV or SV40 (e.g., Mulligan et al.,Nature 277:108-114, 1979); Adenoviridae, such as adenovirus (e.g.,Berkner, Biotechniques 6:616-627, 1988; Rosenfeld et al., Science252:431-434, 1991); Parvoviridae, such as adeno-associated virus (e.g.,Samulski et al., J. Vir. 63:3822-3828, 1989; Mendelson et al., Virol.166:154-165, 1988; PA 7/222, 684); Herpesviridae, such as Herpes SimplexVirus (e.g., Kit, Adv. Exp. Med. Biol. 215:219-236, 1989); andHepadnaviridae (e.g., HBV), as well as certain RNA viruses whichreplicate through a DNA intermediate, such as the Retroviridae (see,e.g., U.S. Pat. No. 4,777,127, GB 2,200, 651, EP 0, 345,242 andWO91/02805; Retroviridae include leukemia in viruses such as MoMLV andimmunodeficiency viruses such as HIV, e.g., Poznansky, J. Virol.65:532-536, 1991).

Similarly, a wide variety of vector systems may be utilized as secondlayer of the eukaryotic layered vector initiation system, including forexample, vector systems derived from viruses of the following families:Picomaviridae (e.g., poliovirus, rhinovirus, coxsackieviruses),Caliciviridae, Togaviridae (e.g., alphavirus, rubella), Flaviviridae(e.g., yellow fever, HCV), Coronaviridae (e.g. HCV, TGEV, IBV, MHV,BCV), Bunyaviridae, Arenaviridae, Retroviridae (e.g., RSV, MoMLV, HIV,HTLV), hepatitis delta virus and Astrovirus. In addition, non-mammalianRNA viruses (as well as components derived therefrom) may also beutilized, including for example, bacterial and bacteriophage replicases,as well as components derived from plant viruses, such as potexviruses(e.g., PVX), carlaviruses (e.g., PVM), tobraviruses (e.g., TRV, PEBV,PRV), Tobamoviruses (e.g., TMV, ToMV, PPMV), luteoviruses (e.g., PLRV),potyviruses (e.g., TEV, PPV, PVY), tombusviruses (e.g., CyRSV),nepoviruses (e.g., GFLV), bromoviruses (e.g., BMV), and topamoviruses.

The replication competency of the autocatalytic vector construct,contained within the second layer of the eukaryotic vector initiationsystem, may be measured by a variety of assays known to one of skill inthe art including, for example, ribonuclease protection assays whichmeasure increases of both positive-sense and negative-sense RNA intransfected cells over time, in the presence of an inhibitor of cellularRNA synthesis, such as dactinomycin, and also assays which measure thesynthesis of a subgenomic RNA or expression of a heterologous reportergene in transfected cells.

Within particularly preferred embodiments of the invention, eukaryoticlayered vector initiation systems are provided that comprise a 5′promoter which is capable of initiating in vivo the synthesis ofalphavirus RNA from cDNA (i.e., a DNA promoter of RNA synthesis),followed by a 5′ sequence which is capable of initiating transcriptionof an alphavirus RNA, a nucleic acid sequence which operably encodes allfour alphaviral nonstructural proteins (including a nucleic acidmolecule as described above which, when operably incorporated into arecombinant alphavirus particle, results in the desired phenotype), analphavirus RNA polymerase recognition sequence, and a 3′ sequence whichcontrols transcription termination/polyadenylation. In addition, a viraljunction region which is operably linked to a heterologous sequence tobe expressed may be included. Within various embodiments, the viraljunction region may be modified, such that viral transcription of thesubgenomic fragment is increased, reduced, or inactivated. Within otherembodiments, a second viral junction region may be inserted followingthe first inactivated viral junction region, the second viral junctionregion being either active or modified such that viral transcription ofthe subgenomic fragment is increased or reduced.

Following in vivo transcription of the eukaryotic layered vectorinitiation system, the resulting alphavirus RNA vector replicon moleculeis comprised of a 5′ sequence which is capable of initiatingtranscription of an alphavirus RNA, a nucleotide sequence encodingbiologically active alphavirus nonstructural proteins, a viral junctionregion, a heterologous nucleotide sequence, an alphavirus RNA polymeraserecognition sequence, and a polyadenylate sequence.

Various aspects of the alphavirus cDNA vector constructs have beendiscussed above, including the 5′ sequence which is capable ofinitiating transcription of an alphavirus, the nucleotide sequenceencoding alphavirus nonstructural proteins, the viral junction region,including junction regions which have been inactivated such that viraltranscription of the subgenomic fragment is prevented, and thealphavirus RNA polymerase recognition sequence. In addition, modifiedjunction regions and tandem junction regions have also been discussedabove.

In another embodiment of the invention, the eukaryotic layered vectorinitiation system is derived from an alphavirus vector, such as aSindbis vector construct, which has been adapted to replicate in one ormore cell lines from a particular eukaryotic species, especially amammalian species, such as humans. For instance, if the gene encodingthe recombinant protein to be expressed is of human origin and theprotein is intended for human therapeutic use, production in a suitablehuman cell line may be preferred in order that the protein bepost-translationally modified as would be expected to occur in humans.This approach may be useful in further enhancing recombinant proteinproduction (as discussed in more detail below). Given the overallplasticity of an alphaviral genome due to the infidelity of the viralreplicase, variant strains with an enhanced ability to establish hightiter productive infection in selected eukaryotic cells (e.g., human,murine, canine, feline, etc.) can be isolated. Additionally, variantalphaviral strains having an enhanced ability to establish high titerpersistent infection in eukaryotic cells may also be isolated using thisapproach. Alphavirus expression vectors can then be constructed fromcDNA clones of these variant strains according to procedures providedherein.

Within another embodiment of the invention, the eukaryotic layeredvector initiation system comprises a promoter for initial alphaviralvector transcription that is transcriptionally active only in adifferentiated cell type. Briefly, it is well established thatalphaviral infection of mammalian cells in culture, such as thosederived from hamster (e.g., baby hamster kidney cells) or chicken (e.g.,chicken embryo fibroblasts), typically results in cytoxicity. Thus, toproduce a stably transformed or transfected host cell line, theeukaryotic layered vector initiation system may be introduced into ahost cell wherein the promoter which enables the initial vectoramplification is a transcriptionally inactive, but inducible, promoter.In a particularly preferred embodiment, such a promoter isdifferentiation state dependent. In this configuration, activation ofthe promoter and subsequent activation of the alphavirus DNA vectorcoincides with induction of cell differentiation. Upon growth to acertain cell number of such a stably transformed or transfected hostcell line, the appropriate differentiation stimulus is provided, therebyinitiating transcription of the vector construct and amplifiedexpression of the desired gene and encoded polypeptide(s). Many suchdifferentiation state-dependent promoters are known to those in the art,as are cell lines which can be induced to differentiate by applicationof a specific stimulus. Representative examples include cell lines F9and P19, HL60, and Freund erythroleukernic cell lines and HEL, which areactivated by retinoic acid, horse serum, and DMSO, respectively.

In a preferred embodiment, such promoters can be regulated by twoseparate components. For example, as described in Example 7, bindingsites for both a transcriptional activator and a transcriptionalrepressor are positioned adjacent to a “core” promoter, in anoperably-dependent manner. In this configuration, the uninduced state ismaintained by blocking the ability of the transcriptional activator tofind its recognition site, while allowing the transcriptional repressorto be constitutively expressed and bound to its recognition site.Induction is permitted by blocking the transcriptional repressor andremoving the transactivator block. For example, atetracycline-responsive promoter system (Gossen and Bujard, Proc. Natl.Acad. Sci. 89:5547-5551, 1992) may be utilized for inducibletranscription of an alphavirus vector RNA. In this system, theexpression of a tetracycline repressor and HSV-VP16 transactivatordomain, as a “fusion” protein (rTA), stimulates in vivo transcription ofthe alphavirus vector RNA by binding specifically to a tetracyclineoperator sequence (tetO) located immediately adjacent to a minimal“core” promoter (for example, CMV). The binding and transactivationevent is reversibly blocked by the presence of tetracycline, and may be“turned on” by removing tetracycline from the culture media. Asuninduced basal levels of transcription will vary among different celltypes, other different minimal core promoters (for example HSV-tk) maybe linked to the tetracycline operator sequences, provided thetranscription start site is known, to allow juxtaposition at or in theimmediate proximity of alphavirus vector nucleotide 1.

The rTA transactivator can be provided by an additional expressioncassette also stably transformed into the same cell line; and in certainembodiments, the rTA expression cassette may itself be autoregulatory.The use of an autoregulatory rTA expression cassette circumventspotential toxicity problems associated with constitutive high levelexpression of rTA by linking expression to transcriptional control bythe same tetO-linked promoter to which rTA itself binds. This type ofsystem creates a negative feedback cycle that ensures very little rTA isproduced in the presence of tetracycline, but becomes highly active whenthe tetracycline is removed. Such an autoregulatory rTA expressioncassette is provided in plasmid pTet-tTAk (Shockett et al., Proc. Natl.Acad. Sci. USA 92:6522-6526, 1995).

For transcriptional repression, the KRAB repression domain of a certainzinc finger proteins can Also be utilized. Briefly, KRAB(Krüppel-associated box) domains are highly conserved sequences presentin the amino-terminal regions of more than one-third of allKrüppel-class Cys₂His₂ zinc finger proteins. The domains contain twopredicted amphipathic α-helicies and have been shown to function as DNAbinding-dependent RNA polymerase II transcriptional repressors (forexample, Licht et al., Nature 346: 76-79, 1990). Like othertranscription factors, the active repression domain and the DNA-bindingdomain are distinct and separable. Therefore, the repression domain canbe linked as a fusion protein to any sequence specific DNA bindingprotein for targeting. Thus, the DNA binding protein component can bereversibly prevented from binding in a regulatable fashion, therebyturning “off” the transcriptional silencing. For example, the KRABdomain from human Kox1 (Thiesen, New Biol. 2:363-374, 1990) can be fusedto the DNA-binding lactose (lac) repressor protein, forming a hybridtranscriptional silencer with reversible, sequence-specific binding to alac operator sequence engineered immediately adjacent to thetet-responsive promoter. In this configuration, constitutive expressionof the lac repressor/KRAB domain fusion (rKR) will result in binding tothe lac operator sequence and the elimination of any “leaky” basaltranscription from the uninduced tet-responsive promoter. When vectorexpression is desired and tetracycline is removed from the system, IPTGis added to prevent rKR-mediated transcriptional silencing.

In addition, KRAB domains from other zinc finger proteins, for example,ZNF133 (Tommerup et al., Hum. Mol. Genet. 2:1571-1575, 1993), ZNF91(Bellefroid et al., EMBO J. 12:1363-1374, 1993), ZNF2 (Rosati et al.,Nucleic Acids Res. 19:5661-5667, 1991), as well as other transferablerepressor domains, for example, Drosophila en or eve genes (Jaynes andO'Farrell, EMBO J. 10:1427-1433, 1991; Han and Manley, Genes Dev.7:491-503, 1993), human zinc finger protein YY1 (Shi et al., Cell67:377-388, 1991), Wilms' tumor suppressor protein WT1 (Madden et al.,Science 253:1550-1553, 1991), thyroid hormone receptor (Baniahmad etal., EMBO J. 11:1015-1023, 1992), retinoic acid receptor (Baniahmad etal., ibid), Kid-1 (Witzgall et al., Proc. Natl. Acad. Sci. USA91:4514-4518, 1994), can likewise be readily used in the gene deliveryvehicles provided herein. Furthermore, the lac repressor/lac operatorcomponent of this system may be substituted by any number of otherregulatable systems derived from other sources, for example, thetryptophan and maltose operons, or GAL4.

E. Recombinant Alphavirus Particles, and Generation and Use of ‘Empty’Togavirus Particles or Togaviruses Particles containing non-homologousviral RNA

Within another aspect of the present invention, the generation ofrecombinant alphavirus particles containing RNA alphavirus vectors,which are capable of infection of eukaryotic target cells, aredescribed. Briefly, such recombinant alphavirus particles generallycomprise one or more alphavirus structural proteins, a lipid envelope,and an RNA vector replicon as described herein.

Methods for generating recombinant alphavirus vector particles may bereadily accomplished by, for example, co-transfection of complementingvector and defective helper (DH) molecules derived from in vitrotranscribed RNA, or, alternatively, plasmid DNA, or by coinfection withvirus (see Xiong et al., Science 243:1188-1191, 1989, Bredenbeek et al.,J. Virol. 67:6439-6446, 1993, Dubensky et al., J. Virol 70:508-519, 1996and Dubensky et al., W/O 95/07994).

Within other aspects, methods for generating recombinant alphavirusvector particles from alphavirus-derived packaging or producer celllines are provided. Briefly, such PCL and their stably transformedstructural protein expression cassettes can be derived using methodsdescribed within W/O 95/07994, or using novel methods described withinthis invention. For example, the production of recombinant alphavirusvector particles by PCL can be accomplished following introduction ofalphavirus-based vector molecules with desirable properties into the PCL(see Example 6), the vectors being derived from in vitro transcribedRNA, plasmid DNA, or previously obtained recombinant alphavirusparticles. In yet a further example, production of recombinant particlesfrom alphavirus vector producer cell lines is described (see Example 7).

Within other embodiments, methods are provided for producing high-titerstable togavirus capsid particles that do not contain any genomic RNA(i.e., contain substantially no viral RNA) or RNA Vector Replicons. Asutilized within the present invention, it should be understood that“substantially no” genomic or RNA Vector Replicon nucleic acids refersto ratios of greater than 10:1, and preferably greater than 15:1 of ³⁵Smethionine versus ³H uridine incorporation into virus particles (ascompared to wild-type) (see, e.g., Example 8 and FIG. 38). For example,within one embodiment empty capsid particles (preferably with the lipidbilayer and lycoprotein complement) are constructed from a selectedpathogenic virus from the togavirus family (such as an Alphavirus orRubivirus), and used as immunogens to establish protective immunityagainst infection with the wild-type togavirus. The empty viralparticles are a desirable immunogenic alternative, as they are unable toreplicate and produce virus, yet are able to generate both cellular andhumoral immune responses. Thus, utilizing the methods which aredescribed in more detail in Example 8, empty capsid particles derivedfrom togaviruses (with or without a lipid bilayer and glycoproteincomplement) can be generated from a wide variety of togaviruses,including, but not limited to. alphaviruses (such as Sindbis Virus(e.g., SIN-1 or wild-type Sindbis virus), Venezuelan Equine Encephalitisvirus, Ross River virus, Eastern Equine Encephalitis virus, WesternEquine Encephalitis virus, and rubiviruses (e.g., rubella),

In a second embodiment, sequences from heterologous viruses which encodepeptides that bind to genomic viral RNA can be inserted into a defectivehelper (DH) expression cassette in the amino terminal region of thealphavirus capsid gene, which has been deleted of the sequences whichencode the region of the protein that binds to the homologous alphavirusgenomic RNA. For example, BHK cells can be electroporated with analphavirus replicon RNA, a DH RNA containing a sequence that encodes aheterologous virus genomic RNA binding peptide, and a replicon derivedfrom the same heterologous virus. Thus, the alphavirus particlesproduced contain a genomic RNA from a heterologous virus, and possessthe host-range tropism of the alphavirus. As one possible example, gagsequences encoding proteins required for retrovirus RNA binding areincluded in the DH expression cassette construct. In this configuration,the resulting alphavirus particles would contain retrovirus vector RNA.

F. Heterologous Sequences

As noted above, a wide variety of nucleotide sequences may be carriedand expressed by the gene delivery vehicles of the present invention.Preferably, the nucleotide sequences should be of a size sufficient toallow production of viable virus. Within the context of the presentinvention the production of any measurable titer by recombinantalphavirus particles, for example, by plaque assay, luciferase assay, orβ-galactosidase assay of infectious virus on appropriate susceptiblemonolayers, or the expression of detectable levels of the heterologousgene product by RNA or DNA vectors, is considered to be “production ofviable virus.” This may be, at a minimum, an alphavirus vector constructwhich does not contain any additional heterologous sequence. However,within other embodiments, the vector construct may contain additionalheterologous or foreign sequences. Within preferred embodiments, theheterologous sequence can comprise a heterologous sequence of at leastabout 100 bases, 2 kb, 3.5 kb, 5 kb, 7 kb, or even a heterologoussequence of at least about 8 kb.

As will be evident to one of ordinary skill in the art given thedisclosure provided herein, the efficiency of recombinant alphavirusparticle packaging and hence, viral titer, is to some degree dependentupon the size of the sequence to be packaged. Thus, in order to increasethe efficiency of packaging and the production of viable virus,additional non-coding sequences may be added to the vector construct.Moreover, within certain embodiments of the invention it may be desiredto increase or decrease viral titer. This increase or decrease may beaccomplished by increasing or decreasing the size of the heterologoussequence, and hence the efficiency of packaging.

As briefly noted above, a wide variety of heterologous sequences may beincluded within the gene delivery vehicles described herein including,for example, sequences which encode palliatives such as lymphokines orcytokines, toxins, prodrug converting enzyme, antigens which stimulatean immune response, ribozymes, proteins for therapeutic application suchas growth or regulatory factors, and proteins which assist or inhibit animmune response, as well as antisense sequences (or sense sequences for“antisense applications”). In addition, as discussed above, the genedelivery vehicles provided herein may contain (and express, withincertain embodiments) two or more heterologous sequences.

1. Lymphokines

Within one embodiment of the invention, the heterologous sequenceencodes a lymphokine. Briefly, lymphokines act to proliferate, activate,or differentiate immune effectors cells. Representative examples oflymphokines include gamma interferon, tumor necrosis factor, IL-1, IL-2,IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13,IL-14, IL-15, GM-CSF, CSF-1 and G-CSF.

Within related embodiments of the invention the heterologous sequenceencodes an immunomodulatory cofactor. Briefly, as utilized within thecontext of the present invention, “immunomodulatory cofactor” refers tofactors which, when manufactured by one or more of the cells involved inan immune response, or when added exogenously to the cells, causes theimmune response to be different in quality or potency from that whichwould have occurred in the absence of the cofactor. The quality orpotency of a response may be measured by a variety of assays known toone of skill in the art including, for example, in vitro assays whichmeasure cellular proliferation (e.g, ³H thymidine uptake), and in vitrocytotoxic assays (e.g., which measure ⁵¹Cr release) (see Warner et al.,AIDS Res. and Human Retroviruses 7:645-655, 1991).

Representative examples of immunomodulatory co-factors include alphainterferon (Finter et al., Drugs 42(5):749-765, 1991; U.S. Pat. No.4,892,743; U.S. Pat. No. 4, 966, 843; WO 85/02862; Nagata et al., Nature284:316-320, 1980; Familletti et al., Methods in Enz. 78:387-394, 198 1;Twu et al., Proc. Natl. Acad Sci. USA 86:2046-2050, 1989; Faktor et al.,Oncogene 5:867-872, 1990), beta interferon (Seif et al., J. Virol.65:664-671, 1991), gamma interferons (Radford et al., American Societyof Hepatology: 2008-2015, 1991; Watanabe et al., PNAS 86:9456-9460,1989; Gansbacher et al., Cancer Research 50:7820-7825, 1990; Maio etal., Can. Immunol. Immunother. 30:34-42, 1989; U.S. Pat. Nos. 4, 762,791 and 4, 727, 138), G-CSF (U.S. Pat. Nos. 4, 999,291 and 4, 810, 643),GM-CSF (WO 85/04188), TNFs (Jayaraman et al., J. Immunology 144:942-951,1990), Interleukin- 2 (IL-2) (Karupiah et al., J Immunology 144:290-298,1990; Weber et al., J. Exp. Med. 166:1716-1733, 1987; Gansbacher et al.,J. Exp. Med. 172:1217-1224, 1990; U.S. Pat. No. 4,738,927), IL-4 (Tepperet al., Cell 57:503-512, 1989; Golumbek et al., Science 254:713-716,1991: U.S. Pat. No. 5,017,691), IL-6 (Brakenhof et al., J. Immunol.139:4116-4121, 1987; WO 90/06370), IL-12, IL-15 (Grabstein et al.,Science 264:965-968, 1994; Genbank-EMBL Accession No. V03099), ICAM-1(Altman et al., Nature 338:512-514, 1989), ICAM-2, LFA-1, LFA-3, MHCclass I molecules, MHC class II molecules, ₂-microglobulin, chaperones,CD3; B7/BB 1, MHC linked transporter proteins or analogues thereof.

The choice of which immunomodulatory cofactor to include within aalphavirus vector construct may be based upon known therapeutic effectsof the cofactor, or experimentally determined. For example, in chronichepatitis B infections alpha interferon has been found to be efficaciousin compensating a patient's immunological deficit and thereby assistingrecovery from the disease. Alternatively, a suitable immunomodulatorycofactor may be experimentally determined. Briefly, blood samples arefirst taken from patients with a hepatic disease. Peripheral bloodlymphocytes (PBLs) are restimulated in vitro with autologous orHLA-matched cells (e.g, EBV transformed cells), and transduced with analphavirus vector construct which directs the expression of animmunogenic portion of a hepatitis antigen and the immunomodulatorycofactor. Stimulated PBLs are used as effectors in a CTL assay with theHLA-matched transduced cells as targets. An increase in CTL responseover that seen in the same assay performed using HLA-matched stimulatorand target cells transduced with a vector encoding the antigen alone,indicates a useful immunomodulatory cofactor. Within one embodiment ofthe invention, the immunomodulatory cofactor gamma interferon isparticularly preferred.

Another example of an immunomodulatory cofactor is the B7/BB 1costimulatory factor. Briefly, activation of the full functionalactivity of T cells requires two signals. One signal is provided byinteraction of the antigen-specific T cell receptor with peptides whichare bound to major histocompatibility complex (MHC) molecules, and thesecond signal, referred to as costimulation, is delivered to the T cellby antigen-presenting cells. The second signal is required forinterleukin-2 (IL-2) production by T cells and appears to involveinteraction of the B7/BB1 molecule on antigen-presenting cells with CD28and CTLA-4 receptors on T lymphocytes (Linsley et al., J. Exp. Med.173:721-730, 1991a, and J. Exp. Med. 174:561-570, 1991). Within oneembodiment of the invention, B7/BB1 may be introduced into tumor cellsin order to cause costimulation of CD8⁺T cells, such that the CD8⁺Tcells produce enough IL-2 to expand and become fully activated. TheseCD8⁺T cells can kill tumor cells that are not expressing B7 becausecostimulation is no longer required for further CTL function. Vectorsthat express both the costimulatory B7/BB1 factor and, for example, animmunogenic HBV core protein, may be made utilizing methods which aredescribed herein. Cells transduced with these vectors will become moreeffective antigen-presenting cells. The HBV core-specific CTL responsewill be augmented from the fully activated CD8⁺T cell via thecostimulatory ligand B7/BB 1.

2. Toxins

Within another embodiment of the invention, the heterologous sequenceencodes a toxin. Briefly, toxins act to directly inhibit the growth of acell. Representative examples of toxins include ricin (Lamb et al., Eur.J. Biochem. 148:265-270, 1985), abrin (Wood et al., Eur. J Biochem.198:723-732, 1991; Evensen et al., J. of Biol. Chem. 266:6848-6852,1991; Collins et al., J. of Biol. Chem. 265:8665-8669, 1990; Chen etal., Fed. of Eur. Biochem Soc. 309:115-118, 1992), diphtheria toxin(Tweten et al., J. Biol. Chem. 260:10392-10394, 1985), cholera toxin(Mekalanos et al., Nature 306:551-557, 1983; Sanchez and Holmgren, PNAS86:481-485, 1989), gelonin (Stirpe et al., J. Biol. Chem. 255:6947-6953,1980), pokeweed (Irvin, Pharmac. Ther. 21:371-387, 1983), antiviralprotein (Barbieri et al., Biochem. J. 203:55-59, 1982; Irvin et al.,Arch. Biochem. & Biophys. 200:418-425, 1980; Irvin, Arch. Biochem. &Biophys. 169:522-528, 1975), tritin, Shigella toxin (Calderwood et al.,PNAS 84:4364-4368, 1987; Jackson et al., Microb. Path. 2:147-153, 1987),Pseudomonas exotoxin A (Carroll and Collier, J. Biol. Chem.262:8707-8711, 1987), herpes simplex virus thymidine kinase (HSVTK)(Field et al., J. Gen. Virol. 49:115-124, 1980), and E. coli. guaninephosphoribosyl transferase.

3. Prodrug converting enzymes.

Within other embodiments of the invention, the heterologous sequenceencodes a prodrug converting enzyme. Briefly, as utilized within thecontext of the present invention, a prodrug converting enzyme refers toa gene product that activates a compound with little or no cytotoxicityinto a toxic product (the prodrug). Representative examples of such geneproducts include HSVTK and VZVTK (as well as analogues and derivativesthereof), which selectively monophosphorylate certain purinearabinosides and substituted pyrimidine compounds, converting them tocytotoxic or cytostatic metabolites. More specifically, exposure of thedrugs ganciclovir, acyclovir, or any of their analogues (e.g., FIAU,FIAC, DHPG) to HSVTK phosphorylates the drug into its correspondingactive nucleotide triphosphate form.

Representative examples of other prodrug converting enzymes which canalso be utilized within the context of the present invention include: E.coli guanine phosphoribosyl transferase which converts thioxanthine intotoxic thioxanthine monophosphate (Besnard et al., Mol. Cell. Biol.7:4139-4141, 1987); alkaline phosphatase, which converts inactivephosphorylated compounds such as mitomycin phosphate anddoxorubicin-phosphate into toxic dephosphorylated compounds; fungal(e.g., Fusarium oxysporum) or bacterial cytosine deaminase, whichconverts 5-fluorocytosine to the toxic compound 5-fluorouracil (Mullen,PNAS 89:33, 1992); carboxypeptidase G2, which cleaves the glutamic acidfrom para-N-bis (2-chloroethyl) aminobenzoyl glutamic acid, therebycreating a toxic benzoic acid mustard; and Penicillin-V amidase, whichconverts phenoxyacetabide derivatives of doxorubicin and melphalan totoxic compounds (see generally, Vrudhula et al., J. of Med. Chem.36(7):919-923, 1993; Kern et al., Canc. Immun. Immunother.31(4):202-206, 1990).

4. Antisense Sequences

Within another embodiment of the invention, the heterologous sequence isan antisense sequence. Briefly, antisense sequences are designed to bindto RNA transcripts, and thereby prevent cellular synthesis of aparticular protein or prevent use of that RNA sequence by the cell.Representative examples of such sequences include antisense thymidinekinase, antisense dihydrofolate reductase (Maher and Dolnick, Arch.Biochem. & Biophys. 253:214-220, 1987; Bzik et al., PNAS 84:8360-8364,1987), antisense HER2 (Coussens et al., Science 230:1132-1139, 1985),antisense ABL (Fainstein et al., Oncogene 4:1477-1481, 1989), antisenseMyc (Stanton et al., Nature 310:423-425, 1984) and antisense ras, aswell as antisense sequences which block any of the cell cycle signalingcomponents (e.g. cyclins, cyclin-dependent kinases, cyclin-dependentkinase inhibitors) or enzymes in the nucleotide biosynthetic pathway. Inaddition, within other embodiments of the invention antisense sequencesto interferon and 2 microglobulin may be utilized in order to decreaseimmune response.

In addition, within a further embodiment of the invention, antisense RNAmay be utilized as an anti-tumor agent in order to induce a potent ClassI restricted response. Briefly, in addition to binding RNA and therebypreventing translation of a specific mRNA, high levels of specificantisense sequences are believed to induce the increased expression ofinterferons (including gamma-interferon) due to the formation of largequantities of double-stranded RNA. The increased expression of gammainterferon, in turn, boosts the expression of MHC Class I antigens.Preferred antisense sequences for use in this regard include actin RNA,myosin RNA, and histone RNA. Antisense RNA which forms a mismatch withactin RNA is particularly preferred.

5. Ribozymes

Within other aspects of the present invention, gene delivery vehiclesare provided which produce ribozymes upon infection of a host cell.Briefly, ribozymes are used to cleave specific RNAs and are designedsuch that it can only affect one specific RNA sequence. Generally, thesubstrate binding sequence of a ribozyme is between 10 and 20nucleotides long. The length of this sequence is sufficient to allow ahybridization with target RNA and disassociation of the ribozyme fromthe cleaved RNA.

A wide variety of ribozymes may be utilized within the context of thepresent invention, including for example, Group I intron ribozymes (Cechet al., U.S. Pat. No. 4, 987, 071); hairpin ribozymes (Hampel et al.,Nucl. Acids Res. 18:299-304, 1990, U.S. Pat. No. 5,254,678 and EuropeanPatent Publication No. 0 360 257), hammerhead ribozymes (Rossi, J. J. etal., Pharmac. Ther. 50:245-254, 1991; Forster and Symons, Cell48:211-220, 1987; Haseloff and Gerlach, Nature 328:596-600, 1988; Walbotand Bruening, Nature 334:196, 1988; Haseloff and Gerlach, Nature334:585, 1988), hepatitis delta virus ribozymes (Perrotta and Been,Biochem. 31:16, 1992); RNase P ribozymes (Takada et al., Cell 35:849,1983); as well as other types of ribozymes (see e.g., WO 95/20241, andWO 95/31551). Further examples of ribozymes include those described inU.S. Pat. Nos. 5, 116, 742, 5,225, 337 and 5,246, 921.

6. Proteins and other cellular constituents

Within other aspects of the present invention, a wide variety ofproteins or other cellular constituents may be carried and/or expressedby the gene delivery vehicles provided herein. Representative examplesof such proteins include native or altered cellular components, as wellas foreign proteins or cellular constituents, found in for example,viruses, bacteria, parasites or fungus.

a. Altered Cellular Components

Within one embodiment, gene delivery vehicles are provided which directthe expression of an immunogenic, non-tumorigenic, altered cellularcomponent (see, e.g., WO 93/10814). As utilized herein, the term“immunogenic” refers to altered cellular components which are capable,under the appropriate conditions, of causing an immune response. Thisresponse must be cell-mediated, and may also include a humoral response.The term “non-tumorigenic” refers to altered cellular components whichwill not cause cellular transformation or induce tumor formation in nudemice. The phrase “altered cellular component” refers to proteins andother cellular constituents which are either associated with rendering acell tumorigenic, or are associated with tumorigenic cells in general,but are not required or essential for rendering the cell tumorigenic.

Briefly, before alteration, the cellular components may be essential tonormal cell growth and regulation and include, for example, proteinswhich regulate intracellular protein degradation, transcriptionalregulation, cell-cycle control, and cell-cell interaction. Afteralteration, the cellular components no longer perform their regulatoryfunctions and, hence, the cell may experience uncontrolled growth.Representative examples of altered cellular components include ras*,p53*, Rb*, altered protein encoded by the Wilms' tumor gene, ubiquitin*,mucin*, protein encoded by the DCC, APC, and MCC genes, the breastcancer gene BRCA1*, as well as receptors or receptor-like structuressuch as neu, thyroid hormone receptor, platelet derived growth factor(PDGF) receptor, insulin receptor, epidermal growth factor (EGF)receptor, and the colony stimulating factor (CSF) receptor.

Once a sequence encoding the altered cellular component has beenobtained, it is necessary to ensure that the sequence encodes anon-tunorigenic protein. Various assays which assess the tumrorigenicityof a particular cellular component are known and may easily beaccomplished. Representative assays include a rat fibroblast assay,tumor formation in nude mice or rats, colony formation in soft agar, andpreparation of transgenic animals, such as transgenic mice.

Tumor formation in nude mice or rats is a particularly important andsensitive method for determining the tumorigenicity of a particularcellular component. Nude mice lack a functional cellular immune system(i.e., do not possess CTLs), and therefore provide a useful in vivomodel in which to test the tumorigenic potential of cells. Normalnon-tumorigenic cells do not display uncontrolled growth properties ifinfected into nude mice. However, transformed cells will rapidlyproliferate and generate tumors in nude mice. Briefly, in one embodimentan alphavirus vector construct is administered to syngeneic murinecells, followed by injection into nude mice. The mice are visuallyexamined for a period of 2 to 8 weeks after injection in order todetermine tumor growth. The mice may also be sacrificed and autopsied inorder to determine whether tumors are present. (Giovanella et al., J.Natl. Cancer Inst. 48:1531-1533, 1972; Furesz et al., Abnormal Cells,New Products and Risk, Hopps and Petricciani (eds.), Tissue CultureAssociation, 1985; and Levenbook et al., J. Biol. Std. 13:135-141,1985.)

Tumorigenicity may also be assessed by visualizing colony formation insoft agar (Macpherson and Montagnier, Virol. 23:291-294, 1964). Briefly,one property of normal non-tumorigenic cells is “contact inhibition”(i.e., cells will stop proliferating when they touch neighboring cells).If cells are plated in a semi-solid agar support medium, normal cellsrapidly become contact inhibited and stop proliferating, whereastumorigenic cells will continue to proliferate and form colonies in softagar.

If the altered cellular component is associated with making the celltumorigenic, then it is necessary to make the altered cellular componentnon-tumorigenic. For example, within one embodiment the sequence or geneof interest which encodes the altered cellular component is truncated inorder to render the gene product non-tumorigenic. The gene encoding thealtered cellular component may be truncated to a variety of sizes,although it is preferable to retain as much as possible of the alteredcellular component. In addition, it is necessary that any truncationleave intact at least some of the immunogenic sequences of the alteredcellular component. Alternatively, multiple translational terminationcodons may be introduced downstream of the immunogenic region. Insertionof termination codons will prematurely terminate protein expression,thus preventing expression of the transforming portion of the protein.

As noted above, in order to generate an appropriate immune response, thealtered cellular component must also be immunogenic. Immunogenicity of aparticular sequence is often difficult to predict, although T cellepitopes often possess an immunogenic amphipathic alpha-helix component.In general, however, it is preferable to determine immunogenicity in anassay. Representative assays include an ELISA, which detects thepresence of antibodies against the newly introduced vector, as well asassays which test for T helper cells such as gamma-interferon assays,IL-2 production assays, and proliferation assays.

As noted above, within another aspect of the present invention, severaldifferent altered cellular components may be co-expressed in order toform a general anti-cancer therapeutic. Generally, it will be evident toone of ordinary skill in the art that a variety of combinations can bemade. Within preferred embodiments, this therapeutic may be targeted toa particular type of cancer. For example, nearly all colon cancerspossess mutations in ras, p53, DCC APC or MCC genes. An alphavirusvector construct which co-expresses a number of these altered cellularcomponents may be administered to a patient with colon cancer in orderto treat all possible mutations. This methodology may also be utilizedto treat other cancers. Thus, an alphavirus vector construct whichco-expresses mucin*, ras*, neu, BRCA1* and p53* may be utilized to treatbreast cancer.

b. Antigens from foreign organisms or other pathogens

Within other aspects of the present invention vectors are provided whichdirect the expression of immunogenic portions of antigens from foreignorganisms or other pathogens. Representative examples of such antigensinclude bacterial antigens (e.g., E. coli, streptococcal,staphylococcal, mycobacterial, etc.), fungal antigens, parasiticantigens, and viral antigens (e.g., influenza virus, Feline LeukemiaVirus (“FeLV”), immunodeficiency viruses such as Feline ImmunodeficiencyVirus (“FIV”) or Human Immunodeficiency Virus (“HIV”), Hepatitis A, Band C Virus (“HAV”, “HBV” and “HCV”, respectively), RespiratorySyncytial Virus, Human Papiloma Virus (“HPV”), Epstein-Barr Virus(“EBV”), Herpes Simplex Virus (“HSV”), Hantavirus, HTLV I, HTLV II andCytomegalovirus (“CMV”). As utilized within the context of the presentinvention, “immunogenic portion” refers to a portion of the respectiveantigen which is capable, under the appropriate conditions, of causingan immune response (i.e., cell-mediated or humoral). “Portions” may beof variable size, but are preferably at least 9 amino acids long, andmay include the entire antigen. Cell-mediated immune responses may bemediated through Major Histocompatability Complex (“MHC”) class Ipresentation, MHC Class II presentation, or both.

Within one aspect of the invention, alphavirus vector constructs areprovided which direct the expression of immunogenic portions ofHepatitis B antigens (see, e.g., WO 93/15207). The Hepatitis B viruspresents several different antigens, including among others, three HB“S” antigens (HBsAgs), an HBc antigen (HBcAg), an HBe antigen (HBeAg),and an HBx antigen (HBxAg) (see Blum et al., TIG 5(5):154-158, 1989).Briefly, the HBeAg results from proteolytic cleavage of a P22 pre-coreintermediate and is secreted from the cell. HBeAg is found in serum as a17 kD protein. The HBcAg is a protein of 183 amino acids, and the HBxAgis a protein of 145 to 154 amino acids, depending on subtype.

The HBsAgs (designated “large, ” “middle” and “small”) are encoded bythree regions of the Hepatitis B genome: S, pre-S2 and pre-S1. The largeprotein, which has a length varying from 389 to 400 amino acids, isencoded by pre-S1, pre-S2, and S regions, and is found in glycosylatedand non-glycosylated forms. The middle protein is 281 amino acids longand is encoded by the pre-S2 and S regions. The small protein is 226amino acids long and is encoded by the S region. It exists in two forms,glycosylated (GP 27^(s)) and non-gllycosylated (P24^(s)). If each ofthese regions are expressed separately, the pre-S1 region will code fora protein of approximately 119 amino acids, the pre-S2 region will codefor a protein of approximately 55 amino acids, and the S region willcode for a protein of approximately 226 amino acids.

As will be evident to one of ordinary skill in the art, variousimmunogenic portions of the above-described S antigens may be combinedin order to induce an immune response when administered by one of thealphavirus vector constructs described herein. In addition, due to thelarge immunological variability that, is found in different geographicregions for the S open reading frame of HBV, particular combinations ofantigens may be preferred for administration in particular geographicregions.

Also presented by HBV are pol (“HBV pol”), ORF 5, and ORF 6 antigens.Briefly, the polymerase open reading frame of HBV encodes reversetranscriptase activity found in virions and core-like particles ininfected livers. The polymerase protein consists of at least twodomains: the amino terminal domain which encodes the protein that primesreverse transcription, and the carboxyl terminal domain which encodesreverse transcriptase and RNase H activity. Immunogenic portions of HBVpol may be determined utilizing methods described herein, utilizingalphavirus vector constructs described below, and administered in orderto generate an immune response within a warm-blooded animal. Similarly,other HBV antigens, such as ORF 5 and ORF 6 (Miller et al., Hepatology9:322-327, 1989) may be expressed utilizing alphavirus vector constructsas described herein.

As noted above, at least one immunogenic portion of an antigen from aforeign organism is incorporated into a gene delivery vehicle. Theimmunogenic portion(s) which are incorporated into the gene deliveryvehicles may be of varying length, although it is generally preferredthat the portions be at least 9 amino acids long and may include theentire antigen. Immunogenicity of a particular sequence is oftendifficult to predict, although T cell epitopes may be predictedutilizing computer algorithms such as TSITES (MedImmune, Md.), in orderto scan coding regions for potential T-helper sites and CTL sites. Fromthis analysis, peptides are synthesized and used as targets in an invitro cytotoxic assay. Other assays, however, may also be utilized,including, for example, ELISA, which detects the presence of antibodiesagainst the newly introduced vector, as well as assays which test for Thelper cells such as gamma-interferon assays, IL-2 production assays andproliferation assays.

Immunogenic portions may also be selected by other methods. For example,the HLA A2.1 transgenic mouse has been shown to be usefull as a modelfor human T-cell recognition of viral antigens. Briefly, in theinfluenza and hepatitis B viral systems, the murine T cell receptorrepertoire recognizes the same antigenic determinants recognized byhuman T cells. In both systems, the CTL response generated in the HLAA2.1 transgenic mouse is directed toward virtually the same epitope asthose recognized by human CTLs of the HLA A2. l haplotype (Vitiello etal., J. Exp. Med. 173:1007-1015, 1991; Vitiello et al., Abstract ofMolecular Biology of Hepatitis B Virus Symposia, 1992).

As noted above, more than one immunogenic portion may be incorporatedinto the gene delivery vehicles. For example, a gene delivery vehiclemay express (either separately or as one construct) all or immunogenicportions of HBcAg, HBeAg, HBsAgs, HBxAg, as well as immunogenic portionsof the HCV antigens C, E1, E2, NS3, NS4, or NS5.

7. Sources for Heterologous Sequences

Sequences which encode the above-described proteins may be readilyobtained from a variety of sources, including for example, depositoriessuch as the American Type Culture Collection (ATCC, Rockville, Md.), orfrom commercial sources such as British Bio-Technology Limited (Cowley,Oxford, England). Representative examples include BBG 12 (containing theGM-CSF gene coding for the mature protein of 127 amino acids); BBG 6(which contains sequences encoding gamma interferon), ATCC No. 39656(which contains sequences encoding TNF), ATCC No. 20663 (which containsequences encoding alpha interferon), ATCC Nos. 31902, 31902 and 39517(which contains sequences encoding beta interferon), ATCC No 67024(which contain a sequence which encodes Interleukin-1b); ATCC Nos.39405, 39452, 39516, 39626 and 39673 (which contains sequences encodingInterleukin-2); ATCC Nos. 59399, 59398, and 67326 (which containsequences encoding Interleukin-3); ATCC No. 57592 (which containssequences encoding Interleukin-4), ATCC Nos. 59394 and 59395 (whichcontain sequences encoding Interleukin-5), and ATCC No. 67153 (whichcontains sequences encoding Interleukin-6).

Sequences which encode altered cellular components as described abovemay be readily obtained from a variety of sources. For example, plasmidswhich contain sequences that encode altered cellular products may beobtained from a depository such as the American Type Culture Collection(ATCC, Rockville, Md.), or from commercial sources such as AdvancedBiotechnologies (Columbia, Md.). Representative examples of plasmidscontaining some of the above-described sequences include ATCC No. 41000(containing a G to T mutation in the 12th codon of ras), and ATCC No.41049 (containing a G to A mutation in the 12th codon).

Alternatively, plasmids which encode normal cellular components may alsobe obtained from depositories such as the ATCC (see, for example, ATCCNo. 41001, which contains a sequence which encodes the normal rasprotein; ATCC No. 57103, which encodes abl; and ATCC Nos. 59120 or59121, which encode the bcr locus) and mutated to form the alteredcellular component. Methods for mutagenizing particular sites mayreadily be accomplished using methods known in the art (see Sambrook etal., supra., 15.3 et seq.). In particular, point mutations of normalcellular components such as ras may readily be accomplished bysite-directed mutagenesis of the particular codon, for example, codons12, 13 or 61.

Sequences which encode the above-described viral antigens may likewisebe obtained from a variety of sources. For example, molecularly viralcloned genes may be obtained from sources such as the American TypeCulture Collection (ATCC, Rockville, Md.). For example. ATCC No. 45020contains the total genomic DNA of hepatitis B (extracted from purifiedDane particles) (see FIG. 3 of Blum et al., TIG 5(5):154-158, 1989) inthe Bam HI site of pBR322 (Moriarty et al., Proc. Natl. Acad. Sci. USA78:2606-2610, 1981).

Alternatively, CDNA sequences which encode the above-describeheterologous sequences may be obtained from cells which express orcontain the sequences. Briefly, within one embodiment, mRNA from a cellwhich expresses the gene of interest is reverse transcribed with reversetranscriptase using oligonucleotide dT or random primers. The singlestranded cDNA may then be amplified by PCR (see U.S. Pat. Nos. 4,683,202; 4, 683, 195 and 4, 800, 159. See also PCR Technology:Principles and Applications for DNA Amplification, Erlich (ed.),Stockton Press, 1989) utilizing oligonucleotide primers complementary tosequences on either side of desired sequences. In particular, adouble-stranded DNA is denatured by heating in the presence of heatstable Taq polymerase, sequence-specific DNA primers, DATP, dCTP, dGTPand dTTP. Double-stranded DNA is produced when synthesis is complete.This cycle may be repeated many times, resulting in a factorialamplification of the desired DNA.

Sequences which encode the above-described proteins may also besynthesized, for example, on an Applied Biosystems Inc. DNA synthesizer(e.g., APB DNA synthesizer model 392 (Foster City, Calif.)).

G. Alphavirus-Packaging/Producer Cell Lines

Within further aspects of the invention, alphavirus packaging andproducer cell lines are provided. In particular, within one aspect ofthe present invention, alphavirus packaging cell lines are providedwherein the viral structural proteins are supplied in trans from one ormore stably transformed expression vectors, and are able to encapsidatetransfected, transduced, or intracellularly produced vector RNAtranscripts in the cytoplasm and release infectious packaged vectorparticles through the cell membrane. In preferred embodiments, thestructural proteins necessary for packaging are synthesized at highlevels only after induction by the RNA vector replicon itself or someother provided stimulus, and the transcripts encoding these structuralproteins are capable of cytoplasmic amplification in a manner that willallow expression levels sufficient to mimic that of a natural viralinfection. Furthermore, in other embodiments, expression of a selectablemarker is operably linked to the structural protein expression cassette.Such a linked selectable marker allows efficient generation offunctional, stably transformed PCL.

For example, alphavirus RNA vector replicon molecules of the desiredphenotype to be packaged, which are themselves capable of autocatalyticreplication in the cell cytoplasm, can be introduced into the packagingcells as in vitro transcribed RNA, recombinant alphavirus particles, oras alphavirus cDNA vector constructs. The RNA vector transcripts thenreplicate to high levels, stimulate amplification of the structuralprotein gene transcript(s) and subsequent protein expression, and aresubsequently packaged by the viral structural proteins, yieldinginfectious vector particles. The intracellular expression of alphavirusproteins and/or vector RNA above certain levels may result in cytotoxiceffects in packaging or producer cell lines. Therefore, within certainembodiments of the invention, it may be desirable for these elements tobe derived from virus variants selected for reduced cytotoxicity oftheir expressed structural proteins, reduced inhibition of hostmacromolecular synthesis, and/or the ability to establish persistentinfection.

To optimize vector packaging cell line performance and final vectortiter, successive cycles of gene transfer and vector packaging may beperformed. For example, supernatants containing infectious packagedvector particles derived from vector transfection of the packaging celllines, can be used to infect or “transduce” a fresh monolayer orsuspension culture of alphavirus packaging cells. Successivetransductions with packaged vector particles and fresh packaging cellsmay be preferred over nucleic acid transfection because of its higherRNA transfer efficiency into cells, optimized biological placement ofthe vector in the cell, and ability to “scale-up” the process for vectorproduction from increasingly larger numbers of packaging cells. Thisleads to higher expression and higher titer of packaged infectiousrecombinant alphavirus vector.

Within other aspects of the invention, a stably integrated or episomallymaintained DNA expression vector can be used to produce the alphavirusvector RNA molecule within the cell. Briefly, such a DNA expressionvector can be configured, in preferred embodiments, to be inducible,such that transcription of the alphavirus vector RNA occurs only whencells have been propagated to a desired density, and are subsequentlyinduced. Once transcribed, the alphavirus vector maintains the abilityto self-replicate autocatalytically and triggers a cascade of eventsthat culminate in packaged vector particle production. This approachallows for continued vector expression over extended periods ofculturing because the integrated DNA vector expression system ismaintained through a drug or other selection marker and the DNA system,once induced, will constitutively express unaltered RNA vector repliconswhich cannot be diluted out by defective RNA copies. Production oflarger-scale, high titer packaged alphavirus vector is possible in thisalphavirus “producer cell line” configuration, the DNA-based alphavirusvector is introduced initially into the packaging cell line bytransfection, since size restrictions could prevent packaging of theexpression vector into a viral vector particle for transduction.

H. Pharmaceutical Compositions

As noted above, the present invention also provides pharmaceuticalcompositions comprising the gene delivery vehicles described herein incombination with a pharmaceutically acceptable carrier, diluent, orrecipient. For example, within one embodiment, RNA or DNA vectorconstructs of the present invention can be lyophilized for long termstorage and transport, and may be reconstituted prior to administrationusing a variety of substances, but are preferably reconstituted usingwater. In certain instances, dilute salt solutions which bring the finalformulation to isotonicity may also be used. In addition, it may beadvantageous to use aqueous solutions containing components whichenhance the activity or physically protect the reconstituted nucleicacid preparation. Such components include cytokines, such as IL-2,polycations, such as protamnine sulfate, lipid formulations, or othercomponents. Lyophilized or dehydrated recombinant vectors may bereconstituted with any convenient volume of water or the reconstitutingagents noted above that allow substantial, and preferably totalsolubilization of the lyophilized or dehydrated sample.

Recombinant alphavirus particles or infectious recombinant virus (bothreferred to as virus below) may be preserved either in crude or purifiedforms. In order to produce virus in a crude form, producing cells mayfirst be cultivated in a bioreactor or flat stock culture, wherein viralparticles are released from the cells into the culture media. Virus maythen be preserved in crude form by first adding a sufficient amount of aformulation buffer to the culture media containing the recombinant virusto form an aqueous suspension. Within certain preferred embodiments, theformulation buffer is an aqueous solution that contains a saccharide, ahigh molecular weight structural additive, and a buffering component inwater. The aqueous solution may also contain one or more amino acids.

The recombinant virus can also be preserved in a purified form. Morespecifically, prior to the addition of the formulation buffer, the cruderecombinant virus described above may be clarified by passing it througha filter and then concentrated, such as by a cross flow concentratingsystem (Filtron Technology Corp., Nortborough, Mass.). Within oneembodiment, DNase is added to the concentrate to digest exogenous DNA.The digest is then diafiltrated in order to remove excess mediacomponents and to establish the recombinant virus in a more desirablebuffered solution. The diafiltrate is then passed over a Sephadex S-500gel column and a purified recombinant virus is eluted. A sufficientamount of formulation buffer is then added to this eluate in order toreach a desired final concentration of the constituents and to minimallydilute the recombinant virus. The aqueous suspension may then be stored,preferably at −70° C., or immediately dried. As above, the formulationbuffer may be an aqueous solution that contains a saccharide, a highmolecular weight structural additive, and a buffering component inwater. The aqueous-solution may also contain one or more amino acids.

Crude recombinant virus may also be purified by ion exchange columnchromatography. Briefly, crude recombinant virus may be clarified byfirst passing it through a filter, followed by loading the filtrate ontoa column containing a highly sulfonated cellulose matrix. Therecombinant virus may then be eluted from the column in purified form byusing a high salt buffer, and the high salt buffer exchanged for a moredesirable buffer by passing the eluate over a molecular exclusioncolumn. A sufficient amount of formulation buffer is then added, asdiscussed above, to the purified recombinant virus and the aqueoussuspension is either dried immediately or stored, preferably at −70° C.

The aqueous suspension in crude or purified form can be dried bylyophilization or evaporation at ambient temperature. Briefly,lyophilization involves the steps of cooling the aqueous suspensionbelow the gas transition temperature or below the eutectic pointtemperature of the aqueous suspension, and removing water from thecooled suspension by sublimation to form a lyophilized virus. Within oneembodiment, aliquots of the formulated recombinant virus are placed intoan Edwards Refrigerated Chamber (3 shelf RC3S unit) attached to a freezedryer (Supermodulyo 12K). A multistep freeze drying procedure asdescribed by Phillips et al. (Cryobiology 18:414, 1981) is used tolyophilize the formulated recombinant virus, preferably from atemperature of −40° C. to −45° C. The resulting composition containsless than 10% water by weight of the lyophilized virus. Oncelyophilized, the recombinant virus is stable and may be stored at −20°C. to 25° C., as discussed in more detail below.

Within the evaporative method, water is removed from the aqueoussuspension at ambient temperature by evaporation. Within one embodiment,water is removed through spray-drying (EP 520, 748). Within thespray-drying process, the aqueous suspension is delivered into a flow ofpreheated gas, usually air, whereupon water rapidly evaporates fromdroplets of the suspension. Spray-drying apparatus are available from anumber of manufacturers (e.g., Drytec, Ltd., Tonbridge, England;Lab-Plant, Ltd., Huddersfield, England). Once dehydrated, therecombinant virus is stable and may be stored at −20° C. to 25° C.Within the methods described herein, the resulting moisture content ofthe dried or lyophilized virus may be determined through use of aKarl-Fischer apparatus (EM Science Aquastar' V1B volumetric titrator,Cherry Hill, N.J.), or through a gravimetric method.

The aqueous solutions used for formulation, as previously described, arepreferably composed of a saccharide, high molecular weight structuraladditive, a buffering component, and water. The solution may alsoinclude one or more amino acids. The combination of these components actto preserve the activity of the recombinant virus upon freezing andlyophilization or drying through evaporation. Although one saccharidethat can be utilized is lactose, other saccharides may likewise beutilized including, for example, sucrose, mannitol, glucose, trehalose,inositol, fructose, maltose or galactose. In addition, combinations ofsaccharides can be used, for example, lactose and mannitol, or sucroseand mannitol. A particularly preferred concentration of lactose is 3%-4%by weight. Preferably, the concentration of the saccharide ranges from1% to 12% by weight.

The high molecular weight structural additive aids in preventing viralaggregation during freezing and provides structural support in thelyophilized or dried state. Within the context of the present invention,structural additives are considered to be of “high molecular weight” ifthey are greater than 5000 m.w. A preferred high molecular weightstructural additive is human serum albumin. However, other substancesmay also be used, such as hydroxyethyl-cellulose,hydroxymethyl-cellulose, dextran, cellulose, gelatin, or povidone. Aparticularly preferred concentration of human serum albumin is 0.1% byweight. Preferably, the concentration of the high molecular weightstructural additive ranges from 0.1% to 10% by weight.

The amino acids, if present, function to further preserve viralinfectivity upon cooling and thawing of the aqueous suspension. Inaddition, amino acids function to further preserve viral infectivityduring sublimation of the cooled aqueous suspension and while in thelyophilized state. A preferred amino acid is arginine, but other aminoacids such as lysine, ornithine, serine, glycine, glutanine, asparagine,glutamic acid or aspartic acid can also be used. A particularlypreferred arginine concentration is 0.1% by weight. Preferably, theamino acid concentration ranges from 0.1% to 10% by weight.

The buffering component acts to buffer the solution by maintaining arelatively constant pH. A variety of buffers may be used, depending onthe pH range desired, preferably between 7.0 and 7.8. Suitable buffersinclude phosphate buffer and citrate buffer. A particularly preferred pHof the recombinant virus formulation is 7.4, and a preferred buffer istromethamine.

In addition, it is preferable that the aqueous solution contain aneutral salt which is used to adjust the final formulated recombinantalphavirus to an appropriate iso-osmotic salt concentration. Suitableneutral salts include sodium chloride, potassium chloride or magnesiumchloride. A preferred salt is sodium chloride.

Aqueous solutions containing the desired concentration of the componentsdescribed above may be prepared as concentrated stock solutions.

It will be evident to those skilled in the art, given the disclosureprovided herein, that it may be preferable to utilize certainsaccharides within the aqueous solution when the lyophilized virus isintended for storage at room temperature. More specifically, it ispreferable to utilize disaccharides, such as lactose or trehalose,particularly for storage at room temperature.

The lyophilized or dehydrated viruses of the subject invention may bereconstituted using a variety of substances, but are preferablyreconstituted using water. In certain instances, dilute salt solutionswhich bring the final formulation to isotonicity may also be used. Inaddition, it may be advantageous to use aqueous solutions containingcomponents known to enhance the activity of the reconstituted virus.Such components include cytokines, such as IL-2, polycations, such asprotamine sulfate, or other components which enhance the transductionefficiency of the reconstituted virus. Lyophilized or dehydratedrecombinant virus may be reconstituted with any convenient volume ofwater or the reconstituting agents noted above that allow substantial,and preferably total solubilization of the lyophilized or dehydratedsample.

I. Methods for Utilizing Gene Delivery Vehicles

As noted above, the present invention also provides methods fordelivering a selected heterologous sequence to a vertebrate (e.g., amammal such as a human or other warm-blooded animal such as a horse,cow, pig, sheep, dog, cat, rat or mouse) or insect, comprising the stepof administering to a vertebrate or insect a gene delivery vehicle asdescribed herein which is capable of expressing the selectedheterologous sequence. Such gene delivery vehicles may be administeredeither directly (e.g., intravenously, intramuscularly,intraperitoneally, subcutaneously, orally, rectally, intraocularly,intranasally), or by various physical methods such as lipofection(Felgner et al., Proc. Natl. Acad. Sci. USA 84:7413-7417, 1989), directDNA injection (Fung et al., Proc. Natl. Acad. Sci. USA 80:353-357, 1983;Seeger et al., Proc. Natl. Acad. Sci. USA 81:5849-5852; Acsadi et al.,Nature 352:815-818, 1991); microprojectile bombardment (Williams et al.,PNAS 88:2726-2730, 1991); liposomes of several types (see, e.g., Wang etal., PNAS 84:7851-7855, 1987); CaPO₄ (Dubensky et al., PNAS81:7529-7533, 1984); DNA ligand (Wu et al, J. Biol. Chem.264:16985-16987, 1989); administration of nucleic acids alone (WO90/11092); or administration of DNA linked to killed adenovirus (Curielet al., Hum. Gene Ther. 3:147-154, 1992); via polycation compounds suchas polylysine, utilizing receptor specific ligands; as well as withpsoralen inactivated viruses such as Sendai or Adenovirus. In addition,the gene delivery vehicles may either be administered directly (i.e., invivo), or to cells which have been removed (ex vivo), and subsequentlyreturned.

As discussed in more detail below, gene delivery vehicles may beadministered to a vertebrate or insect for a wide variety of therapeuticand/or other productive purposes, including for example, for the purposeof stimulating a specific immune response; inhibiting the interaction ofan agent with a host cell receptor: to express a toxic palliative,including for example, conditional toxic palliatives: to immunologicallyregulate the immune system; to prevent cell division, to expressmarkers, for replacement gene therapy, to promote wound healing and/orto produce a recombinant protein. These and other uses are discussed inmore detail below.

1. Immunostimulation

Within one aspect of the present invention, compositions and methods areprovided for administering a gene delivery vehicle which is capable ofpreventing, inhibiting, stabilizing or reversing infectious, cancerous,auto-immune or immune diseases. Representative examples of such diseasesinclude viral infections such as HIV, HBV, HCV, HTLV I, HTLV II, CMV,EBV and HPV, melanomas, diabetes, graft vs. host disease, Alzheimer'sdisease and heart disease. More specifically, within one aspect of thepresent invention, compositions and methods are provided for stimulatingan immune response (either humoral or cell-mediated) to a pathogenicagent, such that the pathogenic agent is either killed or inhibited.Representative examples of pathogenic agents include bacteria, fungi,parasites, viruses and cancer cells.

Within one embodiment of the invention the pathogenic agent is a virus,and methods are provided for stimulating a specific immune response andinhibiting viral spread by using a gene delivery vehicle that directsthe expression of an antigen or modified form thereof to susceptibletarget cells capable of either (1) initiating an immune response to theviral antigen or (2) preventing the viral spread by occupying cellularreceptors required for viral interactions. Expression of the vectornucleic acid encoded protein may be transient or stable with time. Wherean immune response is to be stimulated to a pathogenic antigen, the genedelivery vehicle is preferably designed to express a modified form ofthe antigen which will stimulate an immune response and which hasreduced pathogenicity relative to the native antigen. This immuneresponse is achieved when cells present antigens in the correct manner,i.e., in the context of the MHC class I and/or II molecules along withaccessory molecules such as CD3, ICAM-1, ICAM-2, LFA-1, or analoguesthereof (e.g., Altmann et al., Nature 338:512, 1989). Cells infectedwith gene delivery vehicles are expected to do this efficiently becausethey closely mimic genuine viral infection and because they: (a) areable to infect non-replicating cells, (b) do not integrate into the hostcell genome, (c) are not associated with any life threatening diseases,and (d) express high levels of heterologous protein. Because of thesedifferences, gene delivery vehicles can easily be thought of as safeviral vectors which can be used on healthy individuals for vaccine use.

This aspect of the invention has a further advantage over other systemsthat might be expected to function in a similar manner, in that thepresenter cells are fully viable and healthy, and low levels of viralantigens, relative to heterologous genes, are expressed. This presents adistinct advantage since the antigenic epitopes expressed can be alteredby selective cloning of sub-fragments of the gene for the antigen intothe recombinant alphavirus, leading to responses against immunogenicepitopes which may otherwise be overshadowed by immunodominant epitopes.Such an approach may be extended to the expression of a peptide havingmultiple epitopes, one or more of the epitopes being derived fromdifferent proteins. Further, this aspect of the invention allowsefficient stimulation of cytotoxic T lymphocytes (CTL) directed againstantigenic epitopes, and peptide fragments of antigens encoded bysub-fragments of genes, through intracellular synthesis and associationof these peptide fragments with MHC Class I molecules. This approach maybe utilized to map major inununodominant epitopes for CTL induction.

An immune response may also be achieved by transferring to anappropriate immune cell (such as a T lymphocyte) the gene for thespecific T cell receptor which recognizes the antigen of interest (inthe context of an appropriate MHC molecule if necessary), for animmunoglobulin which recognizes the antigen of interest, or for a hybridof the two which provides a CTL response in the absence of the MHCcontext. Thus, the gene delivery vehicle cells may be used as animmunostimulant, immunomodulator, or vaccine.

In another embodiment of the invention, methods are provided forproducing inhibitor palliatives wherein gene delivery vehicles deliverand express defective interfering viral structural proteins, whichinhibit viral assembly. Such gene delivery vehicles may encode defectivegag, pol, env or other viral particle proteins or peptides and thesewould inhibit in a dominant fashion the assembly of viral particles.This occurs because the interaction of normal subunits of the viralparticle is disturbed by interaction with the defective subunits.

In another embodiment of the invention, methods are provided for theexpression of inhibiting peptides or proteins specific for viralprotease. Briefly, viral protease cleaves the viral gag and gaglpolproteins into a number of smaller peptides. Failure of this cleavage inall cases leads to complete inhibition of production of infectiousretroviral particles. As an example, the HIV protease is known to be anaspartyl protease and these are known to be inhibited by peptides madefrom amino acids from protein or analogues. Gene delivery vehicles toinhibit HIV will express one or multiple fused copies of such peptideinhibitors.

Another embodiment involves the delivery of suppressor genes which, whendeleted, mutated, or not expressed in a cell type, lead to tumorigenesisin that cell type. Reintroduction of the deleted gene by means of a genedelivery vehicle leads to regression of the tumor phenotype in thesecells. Examples of such cancers are retinoblastoma and Wilms Tumor.Since malignancy can be considered to be an inhibition of cellularterminal differentiation compared with cell growth, the alphavirusvector delivery and expression of gene products which lead todifferentiation of a tumor should also, in general, lead to regression.

In yet another embodiment, the gene delivery vehicle provides atherapeutic effect by transcribing a ribozyme (an RNA enzyme) (Haseloffand Gerlach, Nature 334:585, 1989) which will cleave and henceinactivate RNA molecules corresponding to a pathogenic function. Sinceribozymes function by recognizing a specific sequence in the target RNAand this sequence is normally 12 to 17 bp, this allows specificrecognition of a particular RNA species such as a RNA or a retroviralgenome. Additional specificity may be achieved in some cases by makingthis a conditional toxic palliative (see below).

One way of increasing the effectiveness of inhibitory palliatives is toexpress viral inhibitory genes in conjunction with the expression ofgenes which increase the probability of infection of the resistant cellby the virus in question. The result is a nonproductive “dead-end” eventwhich would compete for productive infection events. In the specificcase of HIV, gene delivery vehicles may be delivered which inhibit HIVreplication (by expressing anti-sense tat, etc., as described above) andalso overexpress proteins required for infection, such as CD4. In thisway, a relatively small number of vector-infected HIV-resistant cellsact as a “sink” or “magnet” for multiple nonproductive fusion eventswith free virus or virally infected cells.

2. Blocking Agents

Many infectious diseases, cancers, autoimmune diseases, and otherdiseases involve the interaction of viral particles with cells, cellswith cells, or cells with factors produced by themselves or other cells.In viral infections, viruses commonly enter cells via receptors on thesurface of susceptible cells. In cancers or other proliferativeconditions (e.g., restenosis), cells may respond inappropriately or notat all to signals from other cells or factors, or specific factors maybe mutated, overexpressed, or underexpressed, resulting in loss ofappropriate cell cycle control. In autoimmune disease, there isinappropriate recognition of “self” markers. Within the presentinvention, such interactions may be blocked by producing, in vivo, ananalogue to either of the partners in an interaction. Alternatively,cell cycle control may be restored by preventing the transition from onephase to another (e.g., G1 to S phase) using a blocking factor which isabsent or underexpressed. This blocking action may occurintracellularly, on the cell membrane, or extracellularly, and theaction of an alphavirus vector carrying a gene for a blocking agent, canbe mediated either from inside a susceptible cell or by secreting aversion of the blocking protein to locally block the pathogenicinteraction.

In the case of HIV, the two agents of interaction are the gp 120/gp 41envelope protein and the CD4 receptor molecule. Thus, an appropriateblocker would be a gene delivery vehicle expressing either an HIV envanalogue that blocks HIV entry without causing pathogenic effects, or aCD4 receptor analogue. The CD4 analogue would be secreted and wouldfunction to protect neighboring cells, while the gp 120/gp 41 issecreted or produced only intracellularly so as to protect only thevector-containing cell. It may be advantageous to add humanimmunoglobulin heavy chains or other components to CD4 in order toenhance stability or complement lysis. Administration of a gene deliveryvehicle encoding such a hybrid-soluble CD4 to a host results in acontinuous supply of a stable hybrid molecule. Efficacy of treatment canbe assayed by measuring the usual indicators of disease progression,including antibody level, viral antigen production, infectious HIVlevels, or levels of nonspecific infections.

In the case of uncontrolled proliferative states, such as cancer orrestenosis, cell cycle progression may be halted by the expression of anumber of different factors that affect signaling by cyclins orcyclin-dependent kinases (CDK). For example, the cyclin-dependent kinaseinhibitors, p16, p21, and p27 each regulate cyclin:CDK mediated cellcycle signaling. Overexpression of these factors within a cell by a genedelivery vehicle results in a cytostatic suppression of cellproliferation. Other factors that may be used therapeutically, asblocking agents or targets to disrupt cell proliferation, include, forexample, wild-type or mutant Rb, p53, Myc, Fos, Jun, PCNA, GAX,lentiviral vpr and p15. Within related embodiments, cardiovasculardiseases such as restenosis or atherosclerosis may be treated orprevented with vectors that express products which promotere-endothelialization, or vascular remodeling (e.g., VEGF, TFPI, SOD).

3. Expression of Palliatives

Techniques similar to those described above can be used to produce genedelivery vehicles which direct the expression of an agent (or“palliative”) which is capable of inhibiting a function of a pathogenicagent or gene. Within the present invention, “capable of inhibiting afunction” means that the palliative either directly inhibits thefunction or indirectly does so, for example, by converting an agentpresent in the cells from one which would not normally inhibit afunction of the pathogenic agent to one which does. Examples of suchfunctions for viral diseases include adsorption, replication, geneexpression, assembly, and exit of the virus from infected cells.Examples of such functions for a cancerous cell, cancer-promoting growthfactor, or uncontrolled proliferative condition (e.g., restenosis)include viability, cell replication, altered susceptibility to externalsignals (e.g., contact inhibition), and lack of production or productionof mutated forms of anti-oncogene proteins.

a. Inhibitor Palliatives

In one aspect of the present invention, the gene delivery vehicledirects the expression of a gene which can interfere with a function ofa pathogenic agent, for instance in viral or malignant diseases. Suchexpression may either be essentially continuous or in response to thepresence in the cell of another agent associated either with thepathogenic condition or with a specific cell type (an “identifyingagent”). In addition, vector delivery may be controlled by targetingvector entry specifically to the desired cell type (for instance, avirally infected or malignant cell) as discussed above.

One method of administration is leukophoresis, in which about 20% of anindividual's PBLs are removed at any one time and manipulated in vitro.Thus, approximately 2×10⁹ cells may be treated and replaced. Repeattreatments may also be performed. Alternatively, bone marrow may betreated and allowed to amplify the effect as described above. Inaddition, packaging cell lines producing a vector may be directlyinjected into a subject allowing continuous production of recombinantvirions.

In one embodiment, gene deliver vehicles which express RNA complementaryto key pathogenic gene transcripts (for example, a viral gene product oran activated cellular oncogene) can be used to inhibit translation ofthat transcript into protein, such as the inhibition of translation ofthe HIV tat protein. Since expression of this protein is essential forviral replication, cells containing the gene delivery vehicle would beresistant to HIV replication.

In a second embodiment, where the pathogenic agent is a single-strandedvirus having a packaging signal, RNA complementary to the viralpackaging signal (e.g., an HIV packaging signal when the palliative isdirected against HIV) is expressed, so that the association of thesemolecules with the viral packaging signal will, in the case ofretroviruses, inhibit stem loop formation or tRNA primer bindingrequired for proper encapsidation or replication of the alphavirus RNAgenome.

In a third embodiment, a gene delivery vehicle may be introduced whichexpresses a palliative capable of selectively inhibiting the expressionof a pathogenic gene, or a palliative capable of inhibiting the activityof a protein produced by the pathogenic agent. In the case of HIV, oneexample is a mutant tat protein which lacks the ability to transactivateexpression from the HIV LTR and interferes (in a transdominant manner)with the normal functioning of tat protein. Such a mutant has beenidentified for HTLV II tat protein (“XII Leu⁵” mutant; see Wachsman etal., Science 235:674, 1987). A mutant transrepressor tat should inhibitreplication much as has been shown for an analogous mutant repressor inHSV-I (Friedmann et al., Nature 335:452, 1988).

Such a transcriptional repressor protein can be selected for in tissueculture using any viral-specific transcriptional promoter whoseexpression is stimulated by a virus-specific transactivating protein (asdescribed above). In the specific case of HIV, a cell line expressingHIV tat protein and the HSVTK gene driven by the HIV promoter will diein the presence of ACV. However, if a series of mutated tat genes areintroduced to the system, a mutant with the appropriate properties(i.e., represses transcription from the HIV promoter in the presence ofwild-type tat) will grow and be selected. The mutant gene can then bereisolated from these cells. A cell line containing multiple copies ofthe conditionally lethal vector/tat system may be used to assure thatsurviving cell clones are not caused by endogenous mutations in thesegenes. A battery of randomly mutagenized tat genes are then introducedinto these cells using a “rescuable” alphavirus vector (i.e., one thatexpresses the mutant tat protein and contains a bacterial origin ofreplication and drug resistance marker for growth and selection inbacteria). This allows a large number of random mutations to beevaluated and permits facile subsequent molecular cloning of the desiredmutant cell line. This procedure may be used to identify and utilizemutations in a variety of viral transcriptional activator/viral promotersystems for potential antiviral therapies.

b. Conditional Toxic Palliatives

Another approach for inhibiting a pathogenic agent is to express apalliative which is toxic for the cell expressing the pathogeniccondition. In this case, expression of the palliative from the genedelivery vehicle should be limited by the presence of an entityassociated with the pathogenic agent, such as a specific viral RNAsequence identifying the pathogenic state, in order to avoid destructionof nonpathogenic cells.

In one embodiment of this method, a gene delivery vehicle can beutilized to express a toxic gene (as discussed above) from acell-specific responsive vector. In this manner, rapidly replicatingcells, which contain the RNA sequences capable of activating thecell-specific responsive vectors, are preferentially destroyed by thecytotoxic agent produced by the gene delivery vehicle.

In a similar manner to the preceding embodiment, the gene deliveryvehicle can carry a gene for phosphorylation, phosphoribosylation,ribosylation, or other metabolism of a purine- or pyrimidine-based drug.This gene may have no equivalent in mammalian cells and might come fromorganisms such as a virus, bacterium, fungus, or protozoan. An exampleof this would be the E. coli guanine phosphoribosyl transferase geneproduct, which is lethal in the presence of thioxanthine (see Besnard etal., Mol. Cell. Biol. 7:4139-4141, 1987). Conditionally lethal geneproducts of this type (also referred to as “prodrugs converting enzymes”above) have application to many presently known purine- orpyrimidine-based anticancer drugs, which often require intracellularribosylation or phosphorylation in order to become effective cytotoxicagents. The conditionally lethal gene product could also metabolize anontoxic drug which is not a purine or pyrimidine analogue to acytotoxic form (see Searle et al., Brit. J. Cancer 53:377-384, 1986).

Mammalian viruses in general tend to have “immediate early” genes whichare necessary for subsequent transcriptional activation from other viralpromoter elements. RNA sequences of this nature are excellent candidatesfor activating alphavirus vectors intracellular signals (or “identifyingagents”) of viral infection. Thus, conditionally lethal genes expressedfrom alphavirus cell-specific vectors responsive to these viral“immediate early” gene products could specifically kill cells infectedwith any particular virus. Additionally, since the human and interferonpromoter elements are transcriptionally activated in response toinfection by a wide variety of nonrelated viruses, the introduction ofvectors expressing a conditionally lethal gene product like HSVTK, forexample, in response to interferon production could result in thedestruction of cells infected with a variety of different viruses.

In another aspect of the present invention, gene delivery vehicles areprovided which direct the expression of a gene product capable ofactivating an otherwise inactive precursor into an active inhibitor ofthe pathogenic agent. For example, the HSVTK gene product may be used tomore effectively metabolize potentially antiviral nucleoside analoguessuch as AZT or ddC. The HSVTK gene may be expressed under the control ofa cell-specific responsive vector and introduced into these cell types.AZT (and other nucleoside antivirals) must be metabolized by cellularmechanisms to the nucleotide triphosphate form in order to specificallyinhibit retroviral reverse transcriptase, and thus, HIV replication(Furmam et al., Proc. Natl. Acad. Sci. USA 83:8333-8337, 1986).Constitutive expression of HSVTK (a nucleoside and nucleoside kinasewith very broad substrate specificity) results in more effectivemetabolism of these drugs to their biologically active nucleotidetriphosphate form. AZT or ddC therapy will thereby be more effective,allowing lower doses, less generalized toxicity, and higher potencyagainst productive infection. Additional nucleoside analogues whosenucleotide triphosphate forms show selectivity for retroviral reversetranscriptase but, as a result of the substrate specificity of cellularnucleoside and nucleotide kinases are not phosphorylated, will be mademore efficacious.

Administration of these gene delivery vehicles to human T cell andmacrophage/monocyte cell lines can increase their resistance to HIV inthe presence of AZT and ddC compared to the same cells withoutretroviral vector treatment. Treatment with AZT would be at lower thannormal levels to avoid toxic side effects but still efficiently inhibitthe spread of HIV. The course of treatment would be as described for theblocker.

In one embodiment, the gene delivery vehicle carries a gene specifying aproduct which is not in itself toxic but, when processed or modified bya protein such as a protease specific to a viral or other pathogen, isconverted into a toxic form. For example, the gene delivery vehiclecould carry a gene encoding a proprotein for ricin A chain, whichbecomes toxic upon processing by the HIV protease. More specifically, asynthetic inactive proprotein form of the toxin ricin or diphtheria Achains could be cleaved to the active form by arranging for the HIVvirally encoded protease to recognize and cleave off an appropriate“pro” element.

In another embodiment, the gene delivery vehicle may express a“reporting product” on the surface of the target cells in response tothe presence of an identifying agent in the cells (such as expression ofa viral gene). This surface protein can be recognized by a cytotoxicagent, such as antibodies for the reporting protein, or by cytotoxic Tcells. In a similar manner, such a system can be used as a detectionsystem (see below) to simply identify those cells having a particulargene which expresses an identifying protein.

Similarly, in another embodiment, a surface protein could be expressedwhich would itself be therapeutically beneficial. In the particular caseof HIV, expression of the human CD4 protein specifically in HIV-infectedcells may be beneficial in two ways:

1. Binding of CD4 to HIV env intracellularly could inhibit the formationof viable viral particles, much as soluble CD4 has been shown to do forfree virus, but without the problem of systematic clearance and possibleimmunogenicity, since the protein will remain membrane bound and isstructurally identical to endogenous CD4 (to which the patient should beimmunologically tolerant).

2. Since the CD4/HIV env complex has been implicated as a cause of celldeath, additional expression of CD4 (in the presence of excess HIV-envpresent in HIV-infected cells) leads to more rapid cell death and thusinhibits, viral dissemination. This may be particularly applicable tomonocytes and macrophages, which act as a reservoir for virus productionas a result of their relative refractility to HIV-induced cytotoxicity(which, in turn, is apparently due to the relative lack of CD4 on theircell surfaces).

In another embodiment, the gene delivery vehicle can provide a ribozymewhich will cleave and inactivate RNA molecules essential for viabilityof the vector infected cell. By making ribozyme production dependent ona specific RNA sequence corresponding to the pathogenic state, such asHIV tat, toxicity is specific to the pathogenic state.

4. Exression of Markers

The above-described technique of expressing a palliative in a cell inresponse to a specific RNA sequence can also be modified to enabledetection of a particular gene in a cell which expresses an identifyingprotein (for example, a gene carried by a particular virus), and henceenable detection of cells carrying that virus. In addition, thistechnique enables the detection of viruses (such as HIV) in a clinicalsample of cells carrying an identifying protein associated with thevirus.

This modification can be accomplished by providing a genome coding for aproduct, the presence of which can be readily identified (the “markerproduct”), in a gene delivery vehicle which responds to the presence ofthe identifying protein in the infected cells. For example, HIV, when itinfects suitable cells, makes tat and rev. The indicator cells can thusbe provided with a genome (such as by infection with an appropriaterecombinant alphavirus) which codes for a marker gene, such as thealkaline phosphatase gene, β-galactosidase gene, or the luciferase genewhich is expressed by the recombinant alphavirus upon activation by thetat and/or rev RNA transcript. In the case of β-galactosidase oralkaline phosphatase, exposing the cells to substrate analogues resultsin a color or fluorescence change if the sample is positive for HIV. Inthe case of luciferase, exposing the sample to luciferin will result inluminescence if the sample is positive for HIV. For intracellularenzymes such as β-galactosidase, the viral titre can be measureddirectly by counting colored or fluorescent cells, or by making cellextracts and performing a suitable assay. For the membrane bond form ofalkaline phosphatase, virus titre can also be measured by performingenzyme assays on the cell surface using a fluorescent substrate. Forsecreted enzymes, such as an engineered form of alkaline phosphatase,small samples of culture supernatant are assayed for activity, allowingcontinuous monitoring of a single culture over time. Thus, differentforms of this marker system can be used for different purposes. Theseinclude counting active virus, or sensitively and simply measuring viralspread in a culture and the inhibition of this spread by various drugs.

Further specificity can be incorporated into the preceding system bytesting for the presence of the virus either with or withoutneutralizing antibodies to that virus. For example, in one portion ofthe clinical sample being tested, neutralizing antibodies to HIV may bepresent; whereas in another portion there would be no neutalizingantibodies. If the tests were negative in the system where there wereantibodies and positive where there were no antibodies, this wouldassist in confirming the presence of HIV.

Within an analogous system for an in vitro assay, the presence of aparticular gene, such as a viral gene, may be determined in a cellsample. In this case, the cells of the sample are infected with asuitable gene delivery vehicle which carries the reporter gene which isonly expressed in the presence of the appropriate viral RNA transcript.The reporter gene, after entering the sample cells, will express itsreporting product (such as β-galactosidase or luciferase) only if thehost cell expresses the appropriate viral proteins.

These assays are more rapid and sensitive, since the reporter gene canexpress a greater amount of reporting product than identifying agentpresent, which results in an amplification effect.

5. Immune Down-Regulation

As described above, the present invention also provides gene deliveryvehicles capable of suppressing one or more elements of the immunesystem in target cells infected with the alphavirus. Briefly, specificdown-regulation of inappropriate or unwanted immune responses, such asin chronic hepatitis or in transplants of heterologous tissue such asbone marrow, can be engineered using immune-suppressive viral geneproducts which suppress surface expression of transplantation (MHC)antigen. Group C adenoviruses Ad2 and Ad5 possess a 19 kd glycoprotein(gp 19) encoded in the E3 region of the virus. This gp 19 molecule bindsto class I MHC molecules in the endoplasmic reticulum of cells, andprevents terminal glycosylation and translocation of class I MHC to thecell surface. For example, prior to bone marrow transplantation, donorbone marrow cells may be infected with a gp 19-encoding gene deliveryvehicle which, upon expression of the gp 19, inhibit the surfaceexpression of MHC class I transplantation antigens. These donor cellsmay be transplanted with low risk of graft rejection and may require aminimal immunosuppressive regimen for the transplant patient. This mayallow an acceptable donor-recipient chimeric state to exist with fewercomplications. Similar treatments may be used to treat the range ofso-called autoimmune diseases, including lupus erythromiatis, multiplesclerosis, rheumatoid arthritis or chronic hepatitis B infection.

An alternative method involves the use of anti-sense message, ribozyme,or other specific gene expression inhibitor specific for T cell cloneswhich are autoreactive in nature. These block the expression of the Tcell receptor of particular unwanted clones responsible for anautoimmune response. The anti-sense, ribozyme, or other gene may beintroduced using the viral vector delivery system.

6. Replacement or Augmentation Gene Therapy

One further aspect of the present invention relates to transformingcells of a vertebrate or insect with a gene delivery vehicle whichsupplies genetic sequences capable of expressing a therapeutic protein.Within one embodiment of the present invention, the gene deliveryvehicle is designed to express a therapeutic protein capable ofpreventing, inhibiting, stabilizing or reversing an inherited ornoninherited genetic defect in metabolism, immune regulation, hormonalregulation, enzymatic or membrane associated structural function. Thisembodiment also describes the gene delivery vehicle capable oftransducing individual cells, whereby the therapeutic protein is able tobe expressed systemically or locally from a specific cell or tissue,whereby the therapeutic protein is capable of (a) the replacement of anabsent or defective cellular protein or enzyme, or (b) supplementproduction of a defective of low expressed cellular protein or enzyme.Such diseases may include cystic fibrosis, Parkinson's disease,hypercholesterolemia, adenosine deaminase deficiency, β-globindisorders, Hemophilia A & B, Gaucher's disease, diabetes and leukemia.

As an example of the present invention, a gene delivery vehicle can beconstructed and utilized to treat Gaucher disease. Briefly, Gaucherdisease is a genetic disorder that is characterized by the deficiency ofthe enzyme glucocerebrosidase. This type of therapy is an example of asingle gene replacement therapy by providing a functional cellularenzyme. This enzyme deficiency leads to the accumulation ofglucocerebroside in the lysosomes of all cells in the body. However, thedisease phenotype is manifested only in the macrophages, except in thevery rare neuronpathic forms of the disease. The disease usually leadsto enlargement of the liver and spleen and lesions in the bones. (For areview, see Science 256:794, 1992, and The Metabolic Basis of InheritedDisease, 6th ed.; Scriver et al., vol. 2, p. 1677).

Gene delivery vehicles can similarly be utilized to deliver a widevariety of therapeutic proteins in order to treat, cure, prevent adisease or disease process. Representative examples of such genesinclude, but are not limited to, insulin (see U.S. Pat. No. 4, 431, 740and BE 885196A), hemoglobin (Lawn et al., Cell 21:647-51, 1980),erythropoietin (EPO; see U.S. Pat. No. 4,703,008), megakaryocyte growthand differentiation factor (MGDF), stem cell factor (SCF), G-CSF (Nagataet al., Nature 319:415-418, 1986), GM-CSF, M-CSF (see WO 8706954), theflt3 ligand (Lyman et al. (1993), Cell 75:1157-1167), EGF, acidic andbasic FGF, PDGF, members of the interleukin or interferon families,supra, neurotropic factors (e.g., BDNF; Rosenthal et al., Endocrinology129:1289-1294, 1991, NT-3; see WO 9103569, CNTF; see WO 9104316, NGF;see WO 9310150), coagulation factors (e.g., factors VIII and IX),thrombolytic factors such as t-PA (see EP 292009, AU 8653302 and EP174835) and streptokinase (see EP 407942), human growth hormone (see JP94030582 and U.S. Pat. No. 4, 745, 069) and other animal somatotropins,integrins and other cell adhesion molecules, such as ICAM-1 and ELAM(see also other “heterologous sequences” discussed above), and othergrowth factors, such as IGF-I and IGF-II, TGF-β, osteogenic protein-1(Ozkaynak et al., EMBO J. 9:2085-2093, 1990), and other bonemorphogenetic proteins (e.g., BMP-4, Nakase et al., J. Bone Miner. Res.9:651-659, 1994).

7. Lymphokines and Lymphokine Receptors

As noted above, the present invention also provides gene deliveryvehicles which can, among other functions, direct the expression of oneor more cytokines or cytokine receptors. Briefly, in addition to theirrole as cancer therapeutics, cytokines can have negative effectsresulting in certain pathological conditions. For example, most restingT-cells, B cells, large granular lymphocytes and monocytes do notexpress IL-2R (receptor). In contrast to the lack of IL-2R expression onnormal resting cells, IL-2R is expressed by abnormal cells in patientswith certain leukemias (ATL, Hairy-cell, Hodgkins, acute and chronicgranulocytic), autoimmune diseases, and is associated with allograftrejection. Interestingly, in most of these patients the serumconcentration of a soluble form of IL-2R is elevated. Therefore, withcertain embodiments of the invention therapy may be effected byincreasing the serum concentration of the soluble form of the cytokinereceptor. For example, in the case of IL-2R, a gene delivery vehicle canbe engineered to produce both soluble IL-2R and IL-2R, creating a highaffinity soluble receptor. In this configuration, serum IL-2 levelswould decrease, inhibiting the paracrine loop. This same strategy alsomay be effective against autoimmune diseases. In particular, becausesome autoimmune diseases (e.g., Rheumatoid arthritis, SLE) also areassociated with abnormal expression of IL-2, blocking the action of IL-2by increasing the serum level of receptor may also be utilized in orderto treat such autoimmune diseases.

In other cases inhibiting the levels of IL-1 may be beneficial. Briefly,IL-1 consists of two polypeptides, IL-1 and IL-1, each of which hasplieotropic effects. IL-1 is primarily synthesized by mononuclearphagocytes, in response to stimulation by microbial products orinflammation. There is a naturally occurring antagonist of the IL-1R,referred to as the IL-1 Receptor antagonist (“IL-1Ra”). This IL-1Rantagonist has the same molecular size as mature IL-1 and isstructurally related to it. However, binding of IL-1Ra to the IL-1R doesnot initiate any receptor signaling. Thus, this molecule has a differentmechanism of action than a soluble receptor, which complexes with thecytokine and thus prevents interaction with the receptor. IL-1 does notseem to play an important role in normal homeostasis. In animals,antibodies to IL-1 receptors reduce inflammation and anorexia due toendotoxins and other inflammation inducing agents.

In the case of septic shock, IL-1 induces secondary compounds which arepotent vasodilators. In animals, exogenously supplied IL-1 decreasesmean arterial pressure and induces leukopenia. Neutralizing antibody toIL-1 reduced endotoxin-induced fever in animals. In a study of patientswith septic shock who were treated with a constant infusion of IL-1R forthree days, the 28 day mortality was 16% compared to 44% in patients whoreceived placebo infusions. In the case of autoimmune disease, reducingthe activity of IL-1 reduces inflammation. Similarly, blocking theactivity of IL-1 with recombinant receptors can result in increasedallograft survival in animals, again presumably by decreasinginflammation.

These diseases provide further examples where gene delivery vehicles maybe engineered to produce a soluble receptor or more specifically theIL-1Ra molecule. For example, in patients undergoing septic shock, asingle injection of IL-1Ra producing vector particles could replace thecurrent approach requiring a constant infusion of recombinant IL-1R.

Cytokine responses, or more specifically, incorrect cytokine responsesmay also be involved in the failure to control or resolve infectiousdiseases. Perhaps the best studied example is non-healing forms ofleishmaniasis in mice and humans which have strong, butcounterproductive T_(H)2-dominated responses. Similarly, lepromotomatousleprosy is associated with a dominant, but inappropriate T_(H)2response. In these conditions, gene delivery vehicles may be useful forincreasing circulating levels of IFN gamma, as opposed to thesite-directed approach proposed for solid tumor therapy. IFN gamma isproduced by T_(H)-1 T-cells, and functions as a negative regulator ofT_(H)-2 subtype proliferation. IFN gamma also antagonizes many of theIL-4 mediated effects on B-cells, including isotype switching to IgE.

IgE, mast cells and eosinophils are involved in mediating allergicreaction. IL-4 acts on differentiating T-cells to stimulate T_(H)-2development, while inhibiting T_(H)-1 responses. Thus, alphavirus-basedgene therapy may also be accomplished in conjunction with traditionalallergy therapeutics. One possibility is to deliver a gene deliveryvehicle which produces IL-4R with small amounts of the offendingallergen (i.e., traditional allergy shots). Soluble IL-4R would preventthe activity of IL-4, and thus prevent the induction of a strong T_(H)-2response.

8. Suicide Vectors

One further aspect of the present invention relates to the use of genedelivery vehicle suicide vectors to limit the spread of wild-typealphavirus in the packaging/producer cell lines. Briefly, within oneembodiment the gene delivery vehicle is comprised of an antisense orribozyme sequence specific for the wild-type alphavirus sequencegenerated from an RNA recombination event between the 3′ sequences ofthe junction region of the vector and the 5′ alphavirus structuralsequences of the packaging cell line expression vector. The antisense orribozyme molecule would only be thermostable in the presence of thespecific recombination sequence and would not have any other effect inthe alphavirus packaging/producer cell line. Alternatively, a toxicmolecule (such as those disclosed herein), may also be expressed in thecontext of a vector that would only express in the presence of wild-typealphavirus.

9. Gene Delivery Vehicles to Prevent the Spread of Metastatic Tumors

One further aspect of the present invention relates to the use of genedelivery vehicles for inhibiting or reducing the invasiveness ofmalignant neoplasms. Briefly, the extent of malignancy typically relatesto vascularization of the tumor. One cause for tumor vascularization isthe production of soluble tumor angiogenesis factors (TAF) (Paweletz etal., Crit. Rev. Oncol. Hematol. 9:197, 1989) expressed by some tumors.Within one aspect of the present invention, tumor vascularization may beslowed utilizing gene delivery vehicles to express antisense or ribozymeRNA molecules specific for TAF. Alternatively, anti-angiogenesis factors(Moses et al., Science 248:1408, 1990; Shapiro et al., PNAS 84:2238,1987) may be expressed either alone or in combination with theabove-described ribozymes or antisense sequences in order to slow orinhibit tumor vascularization. Alternatively, gene delivery vehicles canalso be used to express an antibody specific for the TAF receptors onsurrounding tissues.

10. Administration of Gene Delivery Vehicles

Within other aspects of the present invention, methods are provided foradministering a gene delivery vehicle to a vertebrate or insect.Briefly, the final mode of gene delivery vehicle administration usuallyrelies on the specific therapeutic application, the best mode ofincreasing vector potency, and the most convenient route ofadministration. Generally, this embodiment includes gene deliveryvehicles which can be designed to be delivered by, for example, (1)direct injection into the blood stream; (2) direct injection into aspecific tissue or tumor; (3) oral administration; (4) nasal inhalation;(5) direct application to mucosal tissues; or (6) ex vivo administrationof transduced autologous cells into the vertebrate or insect. Withincertain embodiments of the invention, for ex vivo applications cells canbe first removed from a host, positively and/or negatively selected inorder to yield a population of cells which is at least partiallypurified (e.g., CD34⁺stem cells, T cells, or the like), transduced,transfected, or, infected with one of the gene delivery vehicles of thepresent invention, and reintroduced into either the same host or anotherindividual.

Thus, the therapeutic gene delivery vehicle can be administered in sucha fashion such that the vector can (a) transduce a normal healthy celland transform the cell into a producer of a therapeutic protein or agentwhich is secreted systemically or locally, (b) transform an abnormal ordefective cell, transforming the cell into a normal functioningphenotype, (c) transform an abnormal cell so that it is destroyed,and/or (d) transduce cells to manipulate the immune response.

11. Modulation of Transcription Factor Activity

In yet another embodiment, gene delivery vehicles may be utilized inorder to regulate the growth control activity of transcription factorsin the infected cell. Briefly, transcription factors directly influencethe pattern of gene expression through sequence-specifictrans-activation or repression (Karin, New Biologist 21:126-131, 1990).Thus, it is not surprising that mutated transcription factors representa family of oncogenes. Gene delivery vehicles can be used, for example,to return control to tumor cells whose unregulated growth is activatedby oncogenic transcription factors, and proteins which promote orinhibit the binding cooperatively in the formation of homo-andheterodimer trans-activating or repressing transcription factorcomplexes.

One method for reversing cell proliferation would be to inhibit thetrans-activating potential of the c-myc/Max heterodimer transcriptionfactor complex. Briefly, the nuclear oncogene c-myc is expressed byproliferating cells and can be activated by several distinct mechanisms,including retroviral insertion, amplification, and chromosomaltranslocation. The Max protein is expressed in quiescent cells and,independently of c-myc, either alone or in conjunction with anunidentified factor, functions to repress expression of the same genesactivated by the myc/Max heterodimer (Cole, Cell 65:715-716, 1991).

Inhibition of c-myc or c-myc/Max proliferation of tumor cells may beaccomplished by the overexpression of Max in target cells controlled bygene delivery vehicles. The Max protein is only 160 amino acids(corresponding to 480 nucleotide RNA length) and is easily incorporatedinto a gene delivery vehicle either independently, or in combinationwith other genes and/or antisense/ribozyme moieties targeted to factorswhich release growth control of the cell.

Modulation of homo/hetero-complex association is another approach tocontrol transcription factor activated gene expression. For example,transport from the cytoplasm to the nucleus of the trans-activatingtranscription factor NF-B is prevented while in a heterodimer complexwith the inhibitor protein IB. Upon induction by a variety of agents,including certain cytokines, IB becomes phospborylated and NF-B isreleased and transported to the nucleus, where it can exert itssequence-specific trans-activating function (Baeuerle and Baltimore,Science 242:540-546, 1988). The dissociation of the NF-B/IB complex canbe prevented by masking with an antibody the phosphorylation site of IB.This approach would effectively inhibit the trans-activation activity ofthe NF-IB transcription factor by preventing its transport to thenucleus. Expression of the IB phosphorylation site specific antibody orprotein in target cells may be accomplished with an alphavirus genetransfer vector. An approach similar to the one described here could beused to prevent the formation of the trans-activating transcriptionheterodimer factor AP-1 (Turner and Tijan, Science 243:1689-1694, 1989),by inhibiting the association between the jun and fos proteins.

12. Production of Recombinant Proteins

In another aspect of the present invention, togavirus (includingalphavirus) gene delivery vehicles can be utilized to direct theexpression of one or more recombinant proteins in eukaryotic cells (exvivo, in vivo, or established cell lines). As used herein, a“recombinant protein” refers to a protein, polypeptide, enzyme, orfragment thereof. Using this approach, proteins having therapeutic orother commercial application can be more cost-effectively produced.Furthermore, proteins produced in eukaryotic cells may be moreauthentically modified post-translationally (e.g., glycosylated,sulfated, acetylated, etc.), as compared to proteins produced inprokaryotic cells. In addition, such systems may be employed in the invivo production of various chemical compounds, e.g., fine or specialtychemicals.

Within this aspect, the gene delivery vehicle encoding the desiredprotein, enzyme, or enzymatic pathway (as may be required for theproduction of a desired chemical) is transformed, transfected,transduced or otherwise introduced into a suitable eukaryotic cell.Representative examples of proteins which can be produced using such asystem include, but are not limited to, insulin (see U.S. Pat. No.4,431,740 and BE 885196A), hemoglobin (Lawn et al., Cell 21:647-51,1980), erythropoietin (EPO; see U.S. Pat. No. 4,703,008), megakaryocytegrowth and differentiation factor (MGDF), stem cell factor (SCF), G-CSF(Nagata et al., Nature 319:415-418, 1986), GM-CSF, M-CSF (see WO8706954), the flt3 ligand (Lyman et al. (1993), Cell 75:1157-1167), EGF,acidic and basic FGF, PDGF, members of the interleukin or interferonfamilies, supra, neurotropic factors (e.g., BDNF; Rosenthal et al.,Endocrinology 129:1289-1294, 1991, NT-3; see WO 9103569, CNTF; see WO9104316, NGF; see WO 9310150), coagulation factors (e.g., factors VIIIand IX), thrombolytic factors such as t-PA (see EP 292009, AU 8653302and EP 174835) and streptokinase (see EP 407942), human growth hormone(see JP 94030582 and U.S. Pat. No. 4,745, 069) and other animalsomatotropins, integrins and other cell adhesion molecules, such asICAM-1 and ELAM (see also other “heterologous sequences” discussedabove), and other growth factors, such as VEGF, IGF-I and IGF-II, TGF-β,osteogenic protein-1 (Ozkaynak et al., EMBO J. 9:2085-2093, 1990), andother bone or cartilage morphogenetic proteins (e.g., BMP-4, Nakase etal, J. Bone Miner. Res. 9:651-659, 1994). As those in the art willappreciate, once characterized, any gene can be readily cloned into genedelivery vehicles according to the present invention, followed byintroduction into a suitable host cell and expression of the desiredgene. In addition, such vectors may be delivered directly in vivo,either locally or systemically to promote the desired therapeutic effect(e.g., wound healing applications).

Methods for producing recombinant proteins using the vectors andalphavirus packaging cell lines described herein are provided (seeexamples 6 and 7). Briefly, gene delivery vehicles, in the form of invitro transcribed RNA, plasmid DNA, or recombinant vector particles,which encode recombinant proteins, may be introduced (via transfectionor infection) into alphavirus packaging cell lines (PCLs) such that onlya small fraction of the cultured cells (≦1%) contain vector molecules.Vector replicons are packaged by the sPs, supplied in trans by the PCL,following vector RNA amplification, which proceeds according to theSindbis virus replication strategy. In turn, the produced recombinantvector particles infect the remaining cells of the culture. Thus, abloom of recombinant protein expression results over time as recombinantvector particles are produced and subsequently infect all cells in thePCL culture. Similarly, amplification of vector particles with PCL maybe used to generate large, high titer particle stocks for otherapplications. In yet another aspect of this invention, recombinantprotein expression from producer cell lines is described (see Example7). Briefly, cell lines are derived which contain all of the geneticelements, including vector replicon and defective helper expressioncassettes, from which the production of vector particles can be induced,via addition of an extracellular stimulus to the culture. Thus,expression of vector-encoded recombinant protein occurs as a result ofinduction of alphavirus vector particle producer cell lines. In yet astill further aspect of this invention, recombinant protein expressionfrom cell lines stably transformed with eukaryotic layered vectorinitiation systems are described (see Example 7). Briefly, cell linesare derived which are stably transformed with an inducible eukaryoticlayered vector initiation system cassette that encodes a recombinantprotein of interest. Thus, expression of vector-encoded recombinantprotein occurs as a result of induction of the eukaryotic layered vectorinitiation system cassette.

As should be readily understood given the disclosure provided herein,protein production utilizing RNA vectors replicons, eukaryotic layeredvector initiation systems, or recombinant vector particles may also beaccomplished by methods other than introduction into packaging orproducer cell lines. For example, such vectors may be introduced into awide variety of other eukaryotic host cell lines (e.g. COS, BHK, CHO,293, or HeLa cells), as well as direct administration in vivo or to exvivo cells, in order to produce the desired protein.

J. Deposit Information

The following materials have been deposited with the American TypeCulture Collection:

Accession Deposit Designation Deposit Date No. Wild type Sindbis virusCMCC #4639 April 2, 1996 VR-2526 SIN-1 Sindbis virus CMCC #4640 April 2,1996 VR-2527 pBG-SIN1 ELVS1.5 SEAP CMCC #4641 April 2, 1996 97502

The above materials were deposited by Chiron Corporation with theAmerican Type Culture Collection (ATCC), 12301 Parklawn Drive,Rockville, Md. under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for purposesof Patent Procedure. The accession number is available from the ATCC attelephone number (301) 881-2600.

These deposits are provided as convenience to those of skill in the art,and are not an admission that a deposit is required under 35 U.S.C.§112. The nucleic acid sequence of these deposits, as well as the aminoacid sequence of the polypeptides encoded thereby, are incorporatedherein by reference and should be referred to in the event of an errorin the sequence described therein. A license may be required to make,use, or sell the deposited materials, and no such license is grantedhereby.

The following examples are included to more fully illustrate the presentinvention. Additionally, these examples provide preferred embodiments ofthe invention and are not meant to limit the scope thereof. Standardmethods for many of the procedures described in the following examples,or suitable alternative procedures, are provided in widely reorganizedmanuals of molecular biology, such as, for example, “Molecular Cloning,” Second Edition (Sambrook et al., Cold Spring Harbor Laboratory Press,1987) and “Current Protocols in Molecular Biology” (Ausubel et al, eds.Greene Associates/Wiley Interscience, N.Y., 1990).

EXAMPLES Example 1 ISOLATION AND CHARACTERIZATION OF SIN1

Below, the identification and molecular characterization of a positivestrand RNA virus which exhibits reduced inhibition of hostmacromolecular synthesis and is capable of establishing persistentinfection in vertebrate cells, as compared to lytic, cytopathogenic wildtype strains of the same virus, is described. For example, Sindbis virusis used as a prototype representative of the Alphavirus genus.

A. Isolation, plaque purification, and characterization of SIN-1 from awild-type Sindbis virus stock

The isolation, molecular cloning, and characterization of a Sindbisvirus variant strain is described. This strain is able to establishproductive persistent infection in the absence of cytopathicity, butproduce levels of virus equivalent to that of wild-type virus.

A high-titered (>10⁸ PFU/ml) wild-type stock obtained by infection ofBHK cells (ATCC No. CCL-10) with Sindbis virus (CMCC #4639) at low MOI(≦0.1). To facilitate infection, the virus inoculum was contained in avolume just sufficient to cover the monolayer when added to the cells.BHK cells were maintained, and all virus dilutions were performed, inEagle minimal essential medium supplemented with 10% fetal calf serum.Cells were cultured at 37° C. in a 5% CO₂ atmosphere. Extensive CPE, asdemonstrated by “rounding up”, loss of adhesion, and increased lightrefraction of individual cells within the monolayer, and additionally,the decreased overall cell density of the monolayer, was observed within48 hours post infection (hpi). The cell culture fluids were collected,cell debris was removed by low speed centrifugation (4, 000 rpm for 10min at room temperature), and the virus stock was aliquoted and storedat −70° C. The titer of the Sindbis virus stock was determined by plaqueassay as described previously (Strauss et al., Virology 74:154-168,1976). Briefly, chicken embryo fibroblasts (CEF) monolayers wereinfected with various dilutions of the virus stock and the monolayeroverlayed with media supplemented with 0.75% agarose. At 24-48 hpiplaques due to cell lysis were visualized and quantitated eitherdirectly or, alternatively, by staining with crystal violet afterremoving the agarose overlay. The virus titer was determined fromsamples infected with virus dilutions in which the plaques wereaccurately quantitated.

A Sindbis virus stock enriched for DI particles was obtained by repeatedhigh MOI passage (≧5) on BHK cells. BHK monolayers were infectedinitially with the Sindbis virus seed stock at an MOI=5. The culturemedium was collected and clarified by low speed centrifugation aftercomplete cell lysis of the infected culture was observed (usually within24 hpi). The clarified medium collected from the infected culture wasthen used to infect a fresh BHK monolayer. For example, 2 ml of thevirus inoculum was added to a fresh BHK monolayer in a 10 cm petri dish.At 1 hpi, 8 ml of fresh medium was added to the virus-infected culture.As described above, the culture medium was collected and clarified afterobservation of complete cell lysis of the culture. This process wasrepeated until the rate at which cytopathogenicity in the BHK monolayerdeveloped after infection was delayed until at least 4 days. The delayin the onset of cytopathogenicity after infection signifies the presenceof a high level of DI particles in the virus preparation.

The presence of a high level of DI particles in the virus preparationderived from multiple serial undiluted passages of infected cell mediumwas determined by an interference assay or by RNA analysis of BHKinfected cells. In the first method, a homologous interference assay wasperformed as a measure of the presence of DI particles. Briefly, BHKcells were infected alone at an MOI=10 with the high-titered (>10⁸PFU/ml) wild-type stock prepared as described above At 16 hpi, the virusyield was determined by plaque assay, as described above. The virusyield from this experiment was typically 1×10⁹ to 1×10¹⁰ PFU/ml. Inanother experimental group, BHK cells were simultaneously coinfectedwith the wild-type stock (MOI=10) and the virus stock prepared frommultiple serial undiluted passages of infected cell medium (MOI=5). Asbefore, the virus yield was determined at 16 hpi by plaque assay. If thevirus stock from the second experimental group contains a high level ofDI particles, the virus yield will be at least 2-3 orders of magnitudelower than the first experiment (e.g., ≦1×10⁷ PFU/ml)

As a more definitive method for demonstrating the presence of DIparticles in the virus preparation, virus-specific RNA in BHK infectedcells at 16 hpi was analyzed. Briefly, BHK cells were infected (MOI=10)with the high-titered (>10⁸ PFU/ml) wild-type stock or with the virusstock containing DI particles. Mock-infected controls and infected cellswere treated with dactinomycin (1 mg/ml) and labeled with [³H]uridine(20 mCi/ml) from 1 to 16 hpi. RNA was isolated from infected and controlcells by using RNAzol B, as described by the manufacturer (Tel-Test,Inc., Friendswood, Tex.). Alternatively, RNA was isolated withTri-Reagent (Molecular Research Center, Inc., Cincinnati, Ohio), or byconventional methods using phenol extraction of cells lysed in a buffer(0.05 M Tris, 0.1 M NaCl, 0.001 M EDTA, pH 7.5) containing 0.5% Tritonand 0.5% recrystallized naphthalene disulfonate, as described by Weisset al. (J. Virol. 14:1189-1198. 1974). The RNAs were denatured withglyoxal and electrophoresed through 1.1% horizontal agarose gelsprepared in 0.01M sodium phosphate buffer (pH 7.0), at 5V/cm (McMasterand Carmichael, Proc. Natl. Acad. Sci. USA 74:4835-4838, 1977).Alternatively, RNA can be electrophoresed through formaldehyde gels.Following electrophoresis, all moisture was removed from the gels undervacuum with a gel dryer, and the dried gels were treated forfluorography and exposed to film. Two RNAs, corresponding to the genomicand subgenomic species (42S and 26S, respectively), were observed insamples from BHK cells infected with the wild-type virus stock. Incontrast, a large number of RNA species that are distinct from thestandard viral 42S and 26S RNAs were observed in samples from BHK cellsinfected with the virus stock containing DI particles. Multiple RNAscorresponding to DI RNAs migrated predominantly at molecular weightssmaller than the 26S RNA species from wild-type virus. An example ofmultiple RNAs in addition to the 42S and 26S species observed in BHKcells infected with a virus stock containing DI particles may be seen inFIG. 3, lane 3, of Weiss et al. (J. Virol. 33:463-474, 1980).

A Sindbis virus variant strain which is able to establish productivepersistent infection with decreased cytopathogenicity was isolated andmolecularly cloned from a virus stock enriched for DI particles. BHKcells were infected at high multiplicity (MOI=5) with the Sindbis virusstock enriched for DI particles. Cytopathogenicity developed slowlycompared to infection of BHK cells with wild-type virus; however, mostcells were eventually lysed and detached from the plate. Cell debris andnon-adherent cells were removed every two days by medium changes. Withintwo weeks after initial infection, separate and distinct colonies wereobserved. These colonies were thriving and demonstrated no morphologicalevidence of CPE, compared to uninfected BHK cell controls. Within 3-4weeks, the cell colonies were large and discernible to the naked eye.The colonies were isolated with cloning rings, and the cells weredispersed with either 3 mM EDTA or trypsin. Dispersed cells from eachcolony were replated without dilution. Thereafter, cells weresubcultured at a 1:10 dilution upon reaching confluency, generallywithin four days. Aliquots of cells, designated BHK(SIN-1), wereprepared in cryotubes after the fifth passage for long term storage inliquid nitrogen. BHK(SIN-1) cells were indistinguishable from theoriginal, uninfected BHK cells in terms of growth rate or morphology.

B. Molecular Cloning of SIN-1

To characterize the mutation(s) in the Sindbis genome which correlatewith the development of the substantially reduced cytopathogenicity ofSIN-1, genomic RNA from SIN-1 virions was isolated, reverse transcribed,and the resultant cDNA encompassing the nonstructural protein genessequenced, as described more fully below.

Briefly, the SIN-1 virus was plaque purified three times beforepreparation of a stock that was used for the isolation of RNA. TheBHK(SIN-1) cells were grown as described above, the culture fluid wascollected, and various dilutions were used to infect primary chickenembryo fibroblast monolayers (CEF, grown in minimal essential mediumsupplemented with 3% fetal calf serum). Following infection, mediumcontaining 0.5% noble agar was added to the monolayers. Additionally,DEAE-dextran (100 μg/mL) can be included in the agar-overlay medium toincrease the size of the SIN-1 plaques. Individual discreet plaques wereobserved after 3 days of incubation at 30° C. in plates infected withsuitably dilute inoculums of the BHK(SIN-1) culture fluid. After thethird round of purification, the cloned SIN-1 virus was passaged once at30° C. in CEF cells infected at a MOI=0.1. The plaque-purified SIN-1virus preparations were determined to be free of DI particles by theinterference assay and RNA analysis in BHK infected cells, as describedabove.

BHK cells were infected (MOI=10) with the plaque purified SIN-1 stock todetermine the ability of this wild-type Sindbis virus variant strain toestablish persistence. As described above, establishment of persistentinfection in BHK cells with wild-type Sindbis virus requires thepresence of DI particles in the virus preparation and considerable timeto allow those few surviving cells to grow out. In contrast, persistentinfection was readily established in BHK cells infected at 37° C. withthe SIN-1 variant whether or not DI particles are present in the virusinoculum. At six days post infection with SIN-1, the BHK cells werecompletely resistant to superinfection with wild-type Sindbis virus,demonstrating establishment of a persistent infection. However, thesecells were susceptible to infection by the heterologous virus, VSV,demonstrating that interferon is not involved in the establishment ofSIN-1 persistent infection.

CEF cells were infected (MOI=10) with the plaque purified SIN-1 stock togenerate a high titered stock of virus for the isolation of RNA. Ninetyml of culture fluid (2×10¹⁰ PFU/ml) was clarified by centrifugation for5 min at 2, 500 rpm in a Sorvall GSA rotor. The SIN-1 virus was pelletedfrom the clarified culture fluid by centrifugation in an SW27.1 rotorfor 2 h at 24, 000 rpm, at 4° C. The virus pellet was then resuspendedin 3 ml of culture media, homogenized by repeated pipetting, and layeredon top of a 25-40% (w/w) sucrose gradient. This was followed bycentrifugation in an SW27.1 rotor for 4 h at 24, 000 rpm, at 4° C. Thevirus band was visualized with incandescent light illumination, andcollected with a 22 gauge needle/syringe. The RNA was purified furtherby Proteinase K (Boehringer Mannheim, Indianapolis, Ind.) digestion (56°C., 1 hr), followed by extraction with an equal volume of H₂O-saturatedphenol:chlorform (1:1 v/v, pH 7.0), followed by precipitation with 2volumes of ethanol and 0.3 M sodium acetate, pH 5.2.

Alternatively, the SIN-1 virus can be further purified by polyethyleneglycol precipitation of infected BHK cell culture media (Strauss et al.Virology 133:154-168, 1976), or alternatively by pelleting through asucrose cushion (Polo et al. J. Virol. 62:2124-2133, 1988).

Two rounds of first strand cDNA synthesis were performed with thepurified viral RNA, using the Moloney murine leukemia virus orSuperScript II reverse transcriptases (Gibco-BRL, Gaithersburg, Md.)according to the manufacturer's recommended conditions. Six separatereactions were performed, using the Sindbis virus specific primerscomplementary to the positive strand (primers denoted by R) shown below.All primers denoted by R were phosphorylated at their 5′ end withpolynucleotide kinase (New England Biolabs) prior to the first strandsynthesis reaction to facilitate cloning.

Primer Location Seq. ID No. Sequence (5′ -> 3′) Enzyme Site T7/1F SacI/T7/1-20 2 GGTGGAGCTCTAATACGACT Sac I CACTATAGATTGACGGCGTA GTACACAC1465R 1465-1451 3 AATTTCTGCCTCAGC Eco 47 III 1003F 1003-1019 4TATGCAAAGTTACTGAC Eco 47 III 2823R 2823-2806 5 CTGTCATTACTTCATGTC Bsp HI1003F 1003-1019 4 TATGCAAAGTTACTGAC Eco 47 III 4303R 4303-4289 6GCGTGGATCACTTTC Avr II 4051F 4051-4069 7 ATTGCGTGATTTCGTCCGT Avr II8115R 8115-8101 8 TAAATTTGAGCTTTG Pml I 1680F 1680-1689 9GGCATATGGCATTAGTTG Bsp HI 8115R 8115-8101 8 TAAATTTGAGCTTTG Pml I 8034F8034-8052 10 CTGGCCATGGAAGGAAAGG Pml 1 11,703R Xho I/dT₂₁/ 11CCCCTCGAGGGT(21)GAAATG Xho 1 11703-11677 TTAAAAACAAAATTTTGTTG

Synthesis of the second strand from the cDNA template above wasaccomplished in six separate reactions with the Klenow fragment of DNApolymerase I (New England Biolabs, Beverly, Mass.) according to themanufacturer's recommended conditions. Sindbis virus specific primerscomplementary to the negative strand (primers denoted by F shown above)were used. The double-stranded DNA products were substituted, stepwise,for the corresponding regions in the plasmid Toto1101, which containsthe full-length Sindbis virus genome (Rice et al., J. Virol,61:3809-3819, 1987). For example, the T7/1F-1465R product was digestedwith Sac I and Eco 47III, and inserted into Sac I/Eco 47III digested andCIAP treated Toto1101 plasmid, which was purified away from the SacI/Eco 47III small fragment (SP6 promoter, Sindbis virus nts. 1-1407) by1% agarose/TAE (50X/liter: 242 g Tris base/57.1 ml glacial aceticacid/100 ml 0.5 M EDTA pH 8.0) electrophoresis, and GENECLEAN II. Thisconstruct was then digested with Eco 4711I and Avr II (Sindbis virus ntnos. 1407 and 4281, respectively, treated with CIAP, and followed byinsertion of the 3300 bp fragment isolated from the 1003F-4303R product,digested with Eco 47 III and Avr II. The fully assembled clone isdesignated as pRSIN-1g (g, as a reference to full-length genomic clone),and contained all 11, 703 bp of viral genome. A subset of the primerslisted in the table above generate redundant double-stranded DNAreaction products within the SIN-1 genome. For example, the sequences inthe 4051F/8115R product are within the 1680F/8115R product. Theseredundant products are provided as construction alternatives for theSIN-1 genomic clone; ie., in general, the efficiency of cDNA cloning isinversely proportional to the length of the desired fragment.

To clone portions of the viral genome not obtained by the above method,the SIN-1 RNA viral genome was cloned by reverse transcriptionpolymerase chain reaction (RT-PCR). First strand synthesis wasaccomplished as described above. PCR amplifications of Sindbis cDNA withthe primer pairs shown above were performed as separate reactions, usingthe Klentaq1 enzyme, and the reaction conditions, as described in Barnes(Proc. Natl. Acad. Sci. USA 91:2216-2220, 1994). Alternatively, theThermalase thermostable DNA polymerase (Amresco Inc., Solon. Ohio) wassubstituted for the Klentaq 1 enzyme, using a buffer containing 1.5 mMMgCl₂ that was provided by the supplier. Alternatively, the VentRthermostable DNA polymerase (New England Biolabs, Beverly, Mass.) wasused in the amplification reactions. Additionally, the reactionscontained 5% DMSO and “HOT START WAX” beads (Perkin-Elmer>. The PCRamplification protocol used is shown below (The 72° C. extensionincubation period was adjusted to 1 min per 1 kb of template DNA):

Temperature (° C.) Time (Min.) No. Cycles (a) 95 2 1 (b) 95 0.5 55 0.535 72 3.5 (c) 72 10 1

Alternatively, cloning of the full-length SIN-1 RNA genome can beperformed similar to methods which have previously been described(Dubensky et al., J. Virol. 70:508-519, 1996). Briefly, first strandcDNA synthesis is accomplished with a mixture of random hexamer primers(50 ng/ml reaction concentration) (Invitrogen, San Diego, Calif.), andprimer 4B, whose sequence is shown in the table below. Genomic lengthSindbis virus SIN-1 variant cDNA is then amplified by PCR. Six distinctsegments using six pairs of overlapping primers is sufficient to clonethe entire genome. In addition to viral complementary sequences, theSIN-1 5′ end forward primer contains a 19 nucleotide sequencecorresponding to the bacterial SP6 RNA polymerase promoter and the Apa Irestriction endonuclease recognition sequence linked to its 5′ end. Thebacterial SP6 RNA polymerase is poised such that transcription in vitroresults in the inclusion of only a single non-viral G ribonucleotidelinked to the A ribonucleotide, which corresponds to the authenticSindbis virus 5′ end. Inclusion of the Apa I recognition sequencefacilitates insertion of the PCR amplicon into the plasmid vector pKSII⁺(Stratagene, La Jolla, Calif.) polylinker sequence. A five nucleotide‘buffer sequence’ extension is also linked upstream from the Apa Irecognition sequence in order to facilitate efficient enzyme digestion.The sequence of the SP6-5′ SIN-1 forward primer and all of the primerpairs necessary to amplify the entire SIN-1 genome are shown in thetable below. (vote that “nt” and “nts” as utilized hereinafter refer to“nucleotide” and “nucleotides.” respectively). The reference sequence(GenBank accession no. J02363, locus: SINCG) is from Strauss et al.,Virology 133:92-110, 1984.

Recognition Primer Location Seq. ID No. Sequence (5′ -> 3′) SequenceSP6-1A Apa I/SP6/ 12 TATATGGGCCCGATTTAGGTGAC Apa I SIN nts. 1-18ACTATAGATTGACGGCGTAGTAC AC 1B 3182-3160 13 CTGGCAACCGGTAAGTACGATAC Age I2A 3144-3164 14 ATACTAGCCACGGCCGGTATC Age I 2B 5905-5885 15TCCTCTTTCGACGTGTCGAGC Eco RI 3A 5844-5864 16 ACCTTGGAGCGCAATGTCCTG EcoRI 7349R 7349-7328 17 CCTTTTCAGGGGATCCGCCAC Bam HI 7328F 7328-7349 18GTGGCGGATCCCCTGAAAAGG Bam HI 3B 9385-9366 19 TGGGCCGTGTGGTCGTCATG Bcl I4A 9336-9356 20 TGGGTCTTCAACTCACCGGAC Bcl I 10394R 10394-10372 21CAATTCGACGTACGCCTCACTC Bsi WI 10373F 10373-10394 22GAGTGAGGCGTACGTCGAATTG Bsi WI 4B Xba I/T₃₅/ 23 TATATTCTAGA(T₃₅)GAAATGXba I 11703-11698

PCR amplifications of Sindbis cDNA with the primer pairs shown above areperformed as separate reactions, using the Thermalase or Vent_(R) DNApolymerases (cited above), reaction conditions, and the PCRamplification conditions, as described above.

The regions of sequence overlap between the amplification productscorrespond to unique enzyme recognition sites within the PCR amplicon.The PCR products are purified (QIAquick PCR purification kit, Qiagen,Chatsworth, Calif.) and inserted stepwise into the pKS II⁺vector,between the Apa I and Xba I sites. The fully assembled clone isdesignated as pKSRSIN-1 g (g, as a reference to full-length genomicclone), and contains all 11, 703 bp of viral genome.

C. Sequence of the SIN-1 phenotype

The SIN-1 specific nucleotide sequences of the pRSIN-1 g clone wasdetermined by the dideoxy-chain termination method. Sequence comparisonof 8, 000 bp of viral sequence revealed multiple differences between theSIN-1 clone described herein and the Sindbis virus (strain HRsp)sequence provided in GenBank (GenBank Accession no. J02363, locus:SINCG). Differences in the sequence among SIN-1 FIG. 6), SINCG (FIG. 7),and Toto1101 (FIG. 8) are presented below.

SINCG Toto 1101 SIN-1 nt. Position Gene nt aa nt aa nt aa  45 5′ NTR T —T — C —  120 nsP1 C Gln C Gln A Lys 1775 nsP2 G null G null A null 1971nsP2 T Phe T Phe C Leu 2992 nsP2 C Pro T Leu T Leu 3579 nsP2 A Lys G GluG Glu 3855 nsP2 C Pro C Pro T Ser 3866 nsP2 C null C null T null 4339nsP3 A Glu A Glu T Val 4864 nsP3 C Ser C Ser T Phe 5702 nsP3 A null Tnull T null 5854* nsP4 G Arg G Arg A His 7612 junction A — A — T — 7837Capsid C Arg C Arg T Cys *This mutation was found in one cDNA clone. Itwas not detected when the Sin-1 virus RNA was sequenced. It likelyrepresents a minor species in the RNA population.

Verification that the sequence changes were unique to the clone (and notthe result of cloning artifact) described herein, was determined byamplifying SIN-1 virion RNA by RT-PCR as described above, establishingthe sequence containing the nucleotides in question by direct sequencingof the RT-PCR amplicon product, and comparing the sequence to thecorresponding SIN-1 sequence.

D. Characterization and genetic mapping of the SIN-1 phenotype withmolecular clones:

Various regions of the SIN-1 genome were substituted for thecorresponding wild-type Sindbis virus region in the Toto1101 plasmid(Rice et al., J. Virol. 61:3809-3819, 1987) in order to map the locationof the phenotype for establishment of persistence. The various SIN-1 nsPgenes were substituted into the Toto1101 wild-type Sindbis virusbackground using restriction enzyme fragments purified from pRSIN-1 g,as illustrated in the table below.

Restriction Nucleotide nsP Gene Fragment Coordinates Clone DesignationnsP1 Ple I/Eco 47III  98-1407 pRSIN-1nsP1 nsP2 Eco 47III/Avr II1407-4281 pRSIN-1nsP2 nsP2-N terminus Eco 47III/Bgl II 1407-2289pRSIN-1nsP2-N nsP2-C terminus Bgl II/Avr II 2289-4281 pRSIN-1nsP2-CnsP3-4 Avr II/Eco RI 4281-5870 pRSIN-1nsP3 nsP3 Avr II/Spe I 4281-5262pRSIN-1nsP3 nsP4 Spe I/Aat II 5263-5870 pRSIN-1nsP4 nsP1-4 Ple I/Aat II 98-8000 pRSIN-1nsP1-4

The coordinates of the nonstructural gene coding regions are provided inthe following table:

Coordinates of Sindbis nsp Gene virus genome (nt. no.) nsP1  60-1680nsP2 1680-4101 nsP3 4101-5769 nsP4 5769-7597 nsP1-4  60-7597

The various SIN-1, Toto, and chimeric SIN-1/Toto clones, pRSIN-1 g,Toto, pRSIN-1nsP1, pRSIN-1nsP2, pRSIN-1nsP2-N, pRSIN-1nsP2-C,pRSIN-1nsP3, pRSIN-1nsP4, and pRSIN-1nsP1-4 were linearized by digestionwith Xho I, which makes a single cut in the cDNA clones immediatelyadjacent and downstream of a 21 nucleotide poly dA:dT tract followingthe Sindbis virus 3′ end (viral nt. 11703). The linearized clones werepurified with GENECLEAN II (BIO 101, La Jolla, Calif.), and adjusted toa concentration of 0.5 μg/μl. Transcription of the linearized clones wasperformed in vitro at 40° C. for 90 minutes according to the followingreaction conditions: 2 μl DNA/4.25 μl H₂O); 10 μl 2.5 mM NTPs (UTP, ATP,GTP, CTP); 1.25 μl 20 mM Me7G(5′)ppp(5′)G cap analogue; 1.25 μl 100 mMDTT; 5 μl 5× transcription buffer (Promega, Madison Wis.); 0.5 μRNasin(Promega); 0.25 μl 10 μg/μl bovine serum albumin; and 0.5 μl T7 RNApolymerase (Promega). The in vitro transcription reaction products weredigested with DNase I (Promega), purified by sequential phenol:CHCl₃ andether extraction, and followed by ethanol precipitation. Alternatively,the in vitro transcription reaction products can be used directly fortransfection. The in vitro transcription reaction products or purifiedRNA were complexed with a commercial cationic lipid compound(LIPOFECTIN, GIBCO-BRL, Gaithersburg, Md.) and applied to Baby HamsterKidney-21 (BHK-21) cells maintained in a 60 mm petri dish at 75%confluency. Alternatively, BHK cells were electroporated with the invitro transcription reaction products or purified RNA, exactly asdescribed previously (Liljestrom, Bio/Technology 9:1356-1361, 1991). Thetransfected cells were incubated at 37° C. At 48 hourspost-transfection, culture media were collected and the titer of eachvirus was determined by plaque assay, as described above. The titeredvirus stocks derived from these in vitro transcription reactions weredesignated as shown in the table below.

Clone Designation Virus Designation pRSIN-1nsP1 SIN-1nsP1 pRSIN-1nsP2SIN-1nsP2 pRSIN-1nsP2-N SIN-1nsP2-N pRSIN-1nsP2-C SIN-1nsP2-CpRSIN-1NSp3-4 SIN-1nsP3-4 pRSIN-1nsP3 SIN-1nsP3 pRSIN-1nsP4 SIN-1nsP4pRSIN-1nsP1-4 SIN-1nsP1-4 Toto 1101 Toto

To map the SIN-1 persistent phenotype, 8×10⁵ BHK cells were infected(MOI=5) with each of the virus stocks prepared above. At 3 days postinfection, the culture viability was determined by trypan blue dyeexclusion. The results of this experiment (shown below), demonstratethat the SIN-1 phenotype of establishing persistent non-cytocidalinfections maps to the nonstructural genes, and to nsP2 gene inparticular. The number of cells in the mock-infected culture representscontinued growth of these cells until they reached the stationary phase.At 3 dpi, cells infected with SIN-1nsP1, SIN-1nsP3, SIN-1-nsP3-4 andSIN-1nsP4 had all died. The cells that survived infection with SIN-1nsP2and SIN-1nsP1-4 continued to grow and were persistently infected basedon staining with antibodies specific for Sindbis virus.

Virus Number of Cells at 3 dpi SIN-1nsP1 0 SIN-1nsP2 3 × 10⁵ SIN-1nsP3-40 SIN-1nsP3 0 SIN-1nsP4 0 SIN-1nsP1-4 5 × 10⁵ Toto 0 Mock 1 × 10⁷

As shown above, the observed SIN-1 phenotype of establishingnon-cytocidal persistent infections maps to the viral nsPs, as opposedto the sPs. This conclusion was demonstrated clearly by comparison ofcell survival levels between cultures infected with the Toto or SIN-1nsP1-4 virus stocks. Both the Toto and SIN-1 nsP1-4 viruses contain thewild-type sPs; cell survival was observed, however, only in thosecultures infected with the virus (SIN-1nsP1-4) containing nsPs derivedfrom the SIN-1 clone. In these experiments, cell survival was notdependent upon the source of the Sindbis virus sPs. Importantly, theSIN-1 phenotype was mapped further to nsP2. The level of cell survivalwas comparable between cultures infected with the SIN-1nsP1-4 orSIN-1nsP2 viruses. Further, a C→T transition at nucleotide 3855, in theSIN-1 nsP2 gene is responsible for the characteristic phenotype ofestablishment of persistent infection in cells infected with the SIN-1virus. The single proline to serine change in the nsP2 protein producedin cells infected with the chimeric virus SIN-1nsP2-C, was all that wasrequired to convert wild-type Sindbis virus (Toto 1101) from a virusthat killed all of the infected cells into a virus which permitted manyof the infected cells to survive and continue to produce virus. Thephenotypes of chimeric viruses derived from insertion of the SIN-1 nsP1,nsP3, or nsP4 genes into the Toto background were indistinguishable fromwild-type and complete lysis was observed in cultures infected withthese viruses.

The possible effect of amino acid changes in the SIN-1 nsps on the levelof productive infection in BHK cells was determined by comparing thevirus yield over a time course in BHK cells inoculated with the variousSIN-1, wild-type (Toto), or SIN-1/Toto chimeric strains. Briefly, BHKcells were infected (MOI=20) with SIN-1 (plaque purified stock describedabove), Toto, SIN-1nsP1-4, or SIN-1 nsP2 viruses, and the culture fluidswere collected at 3, 6, 9, and 12 hours post infection. The titers ofvirus in the culture fluids were then determined by plaque assay, asdescribed above. The results of this study, shown in FIG. 2, demonstratethat equivalent levels of virus were produced in BHK cells infected withwild-type or SIN-1 strains. The actual virus titers at the 12 hpi timepoint are set forth in the table below. More than half of the BHK cellssurvived infection with SIN-1 virus (and chimeric viruses containingSIN-1 nsPs1-4, or SIN-1 nsP2) in combination with levels of virusproduction equivalent to wild-type strains.

Virus Titer (PFU/ml) at 12 hpi (×10⁹) SIN-1 3.6 Toto 2.7 SIN-1nsP1-4 1.8SIN-1nsP2 2.6

The possible effect of amino acid changes in the SIN-1 nsPs on the levelof viral-specific RNA synthesis was determined by comparing the level of[³H]-uridine incorporation over a time course in BHK cells inoculatedwith the various SIN-1, wild-type (Toto), or SIN-1/Toto chimericstrains. BHK cells (3×10⁵ cells/35 mm dish) were grown at 37° C.,according to the conditions described herein. The cells were infected(MOI=20) with SIN-1, Toto1101, SIN-1nsP1-4, or SIN-1nsP2 viruses. At 30min. post infection, the culture medium was adjusted to 1 μg/mlactinomycin D. After incubation for an additional 30 min, the culturemedium was adjusted to 10 μCi/ml [³H]-uridine. At 3, 6, 9, and 12 hpi,the treated cells were washed with PBS, and lysed by addition of 200 μlof TTE buffer (0.2% Triton X-100, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA).The RNA was precipitated at 4° C. by addition of 200 μl of a 25%trichloroacetic acid (TCA) solution. The RNA was pelleted bymicrocentrifugation for 5 min at 14, 000 rpm, rinsed once with 5% TCA,and dissolved in a solution consisting of 50 mM NaOH/0.1% SDS, at 55° C.The solution containing the dissolved RNA was transferred intoscintillation vials and the level of incorporated [³H]-uridine wasdetermined using EcoLume (ICN, Irvine, Calif.) scintillation fluid. Theresults of this study, shown in FIG. 3, demonstrate that levels ofvirus-specific RNA were dramatically lower in BHK cells infected withSIN-1 compared to wild-type virus. This phenotype of low level ofvirus-specific RNA synthesis maps to the nsPs, as shown by theequivalently low levels of RNA produced in BHK cells infected with theSIN-1 or SIN-1nsP 1-4 strains, compared to wild-type.

Virus [³H]uridine incorporation (×10³ cpm) SIN-1 22 Toto 1101 86SIN-1nsP1-4 28 SIN-1nsP2 62 Mock 8

Virus-specific RNA synthesis in infected BHK cells was also determinedusing all of the SIN-1, Toto, and SIN-1/Toto chimeric, strains. Thelevels of [³H]uridine incorporation, relative to wild-type infection(Toto) at 9 hpi, are shown in FIG. 4, and given in the table below.

Relative RNA Standard Virus No. of Experiments Synthesis Level DeviationToto 1101 9 1.0 SIN-1 9 0.1 ±0.1 SIN-1nsP1-4 8 0.2 ±0.1 SIN-1nsP1 7 1.0±0.2 SIN-1nsP2 8 0.6 ±0.1 SIN-1nsP3 4 1.0 ±0.0 SIN-1nsP3-4 7 0.4 ±0.1SIN-1nsP4 6 0.8 ±0.1 SIN-1nsP2-N 1 0.9 SIN-1nsP2-C 3 0.6 ±0.2 Mock 9 0.0

Thus, BHK cells survive infection with SIN-1 virus (and chimeric virusescontaining SIN-1 nsPs1-4, or SIN-1 nsP2), SIN-1 virus levels equivalentto wild-type strains are produced in BHK cells, and, the level ofviral-specific RNA synthesized is 10-fold lower compared to wild-typevirus.

The possible effect of amino acid changes in the SIN-1 nsPs on the levelof inhibition of host cell protein synthesis was determined by comparingthe level of protein synthesis, relative to uninfected cells, over atime course in BHK cells inoculated with the various SIN-1, wild-type(Toto), or SIN-1/Toto chimeric strains. Briefly, 35 mm dishes seededwith 2×10⁵ BHK cells were infected (MOI=20) with the various Sindbisvirus strains in a 0.5 ml virus inoculum in a buffer consisting ofPBS/1% fetal calf serum. The plates were incubated at 4° C. for 1 hrwith continuous gentle shaking. The inoculum was replaced with 2 ml ofmedium, described previously, containing 10% fetal calf serum, and thedishes were placed in a CO₂ incubator at 37° C. At 5, 8, and 11 hrlater, the media was replaced with 2 ml of MEM lacking methionine(Met-), with 2% fetal calf serum, and incubated 30 min. The medium wasthen replaced with 1 ml of MEM (Met-) containing 2% fetal calf serum and10 μCi/ml of [³⁵S]methionine. Following a 30 min incubation period at37° C., 1 ml of medium containing 10% fetal calf serum was added to eachwell, and the dishes were incubated for another 30 min at 37° C. Thisdilution is sufficient to inhibit further incorporation of radioactivelabel into protein and significantly decreases the background of free[³⁵S]-methionine detected in polyacrylamide gels. The medium was thenremoved from the well. Cells were washed three times with PBS, scrapedfrom the dish into PBS, pelleted by centrifugation, and dissolved in 25μl of loading buffer (0.06 M Tris-HCl, pH 6.7, 2% SDS, 5%β-mercaptoethanol, 5% glycerol, 0.05% bromophenol blue). One-fifth ofthe sample was analyzed on the gel. After electrophoresis, the gels werestained with Coomassie brilliant blue R, dried, and autoradiographed.The rates of inhibition of host cell protein synthesis were compared byquantitating the amount of radioactivity in the section of the gelcontaining only host proteins. The results of this study are shown inFIG. 5, and demonstrate that the level of inhibition of host cellprotein synthesis is significantly lower in SIN-1 virus infected cells,compared to wild-type virus infected cells, particularly at the earlier6 and 9 hour time points post infection.

In summary, BHK cells survive SIN-1 infection, virus levels equivalentto wild-type strains are produced in BHK cells, the level ofviral-specific RNA synthesized is 10-fold lower than wild-type virus,and the level of inhibition of host cell protein synthesis in SIN-1virus infected cells is significantly lower compared to wild-type virusinfected cells. The phenotypes of the SIN-1 virus described herein mapto the viral nsP genes.

Example 2 Isolation and Characterization of Positive Strand RNA VirusesWhich Exhibit Reduced Inhibition of Host Macromolecular Synthesis

The derivation of virus variants exhibiting the desired phenotypes ofreduced, delayed or no inhibition of host cell macromolecular synthesisis dependent on the generation, characterization, and isolation ofsequences which differ from that of wild-type virus. However, inaddition to example 1, there are no obvious or previously disclosedmethods to select for or identify coding or non-coding viral sequencechanges that result in alteration of this virus-based inhibition ofmacromolecular synthesis, or the generation of viruses that lead topersistent, rather than lytic, infection. The present invention providesspecific methods, using alphaviruses as an example, that enable one toovercome these obstacles.

A. Biological Selection of Virus Variants

The biological derivation of virus variants which result in reduced,delayed, or no inhibition of host macromolecular synthesis, or whichestablish persistent infections, can be performed by allowing fornatural, spontaneous mutation within a cell, or first subjecting thedesired virus stock to physical, chemical or other artificialmutagenesis, followed by infection of susceptible cells, and successiveenrichments for those cell populations which harbor mutated virus. It ispossible that prior mutagenesis, although not required, will facilitatethe generation of appropriate mutations. The selection is based on theability of cells infected with the desired variant to survive forsignificantly longer periods than wild-type virus infected cells. Thefollowing examples provide representative methods in detail, usingSindbis virus as an example; however, other viruses are readilysubstituted, as noted in the detailed description.

Specifically, in the case of chemical mutagenesis, a Sindbis virus stocksuspension with a titer of greater than or equal to 10⁹ pfu/ml istreated with nitrosoguanidine at a final concentration of 100 ug/ml.After 15 minutes at room temperature, the nitrosoguanidine is removed bydialysis at 4° C. and the mutagenized stock is subsequently used forinfection. Approximately 5×10⁶ cells of the desired type (for exampleBHK-21), grown in flat stock culture, are infected with the mutagenizedvirus at a multiplicity of infection (M.O.I.) of approximately 5, toensure that every cell is infected. At 12,24, 36, and 48 hourspost-infection, the cell monolayer is washed twice with fresh media toremove dead cells, and replaced with media consisting of a mixture of50% fresh media and 50% conditioned media. After a desired timepost-infection (for example 72 hours), the remaining cells are gentlytrypsinized to detach them from the culture dish and strip away anycell-associated extracellular virus, which is then separated from thecells by differential centrifugation. The remaining cells are thenre-seeded directly into a tissue culture dish containing asemi-confluent uninfected cell monolayer, at a ratio of 1 infected cellfor every 10³ uninfected cells. Additional rounds of selection areperformed by seeding onto uninfected cells for amplification, followedby the above washing and harvesting steps. Alternatively, the initialinfections may be done using a wild-type virus stock at low M.O.I.,allowing for spontaneous mutation during replication within the cell.Using this approach, a heterogenous population of mutant virus isproduced by the infected cells. In those instances where the populationof infected cells recovered after trypsinization includes a significantnumber of non-viable or severely damaged cells, a brief treatment (5minutes at room temperature) with 0.75% NH₄Cl in 17 mM Tris (pH 7.65with HCl), or centrifugation through Percoll™, is included to removeremaining dead or damaged cells, prior to re-seeding onto uninfectedcell monolayers. After a minimum of two successive rounds of selection,virus variants displaying the desired phenotype are isolated by limitingdilution or plaque purification, and subjected to cDNA cloning asdescribed in Example 1. Isolation and characterization of specificsequences responsible for the variant phenotype are accomplished bysubstitution of defined regions of cDNA into a genomic clone orexpression vector and testing for the accompanying phenotypic change, asoutlined in the Examples.

B. Genetic Selection of Virus Variants

In a related approach, natural mutation or random mutagenesis isperformed, not on a virus stock, but rather, using cloned genomic cDNAof the virus that can be transcribed into infectious viral RNA in vitroor in vivo. For example, in the case of prior mutagenesis, plasmidpRSINg, which contains a full-length genomic Sindbis cDNA functionallylinked to a bacteriophage SP6 promoter (Dubensky, et al., J. Virol.70:508-519, 1996), is transformed into competent E. coli XL1-Red mutatorcells and plated on ampicillin plates to obtain colonies. At least 200colonies are chosen at random, pooled, and inoculated for overnightgrowth in a 10 ml broth culture containing ampicillin. Plasmid DNA isprepared from the culture to obtain a heterogeneous population of pRSINgharboring various mutations. The DNA is linearized with Xba I andtranscribed in vitro using SP6 polymerase, as described previously (Riceet al., J. Virol. 61:3809-3819, 1987). RNA transcripts are subsequentlytransfected into the desired cell type (for example, BHK cells) byelectroporation (Liljestrom and Garoff, Bio/Technology 9:1356-1361,1991), for initiation of the Sindbis virus infection cycle.Alternatively, in vitro transcribed RNA from an unmutagenized templatealso may be transfected. Selection of virus mutants which establish apersistent infection or exhibit reduced, delayed, or no inhibition ofhost macromolecular synthesis is performed as above. The selected virusvariants with the desired phenotype are isolated by limiting dilution orplaque purification, subjected to cDNA cloning, and the sequencesresponsible for the variant phenotype are isolated and characterized, asdescribed previously.

C. Genetic Selection of Variants Using Virus-Derived Vectors

1. Vectors Expressing an Immunogenic Protein

In another approach, spontaneous intracellular mutation, or randommutagenesis is performed on virus-derived sequences of a viral-basedexpression vector. These sequences include non-coding and regulatoryregions, as well as nonstructural protein encoding regions. In certaininstances, structural protein-encoding sequences also may be included.Such random mutagenesis or spontaneous intracellular mutation may beperformed using any of the techniques described in this invention, alongwith the cloned cDNA of a virus-derived vector which can be transcribedinto RNA in vitro or in vivo. For example, a replication-competentSindbis virus expression vector may be used to express an immunogeniccell surface protein or other peptide which may be bound by specificantibodies added to the infected cells. Cells which contain functionalvector are identified by their expression of the vector-encodedheterologous antigen and ability to be bound by antibody specific forthe encoded antigen. By limiting the selection process to cellssurviving for extended periods (see above), only those harboring vectorvariants exhibiting the desired phenotype are enriched.

Specifically, in the case of random mutagenesis, plasmid pTE3′2J (Hahnet al., J. Virol.89:2679-2683, 1992), comprising an SP6 promoteroperably linked to a full-length genomic Sindbis cDNA with a duplicatedsubgenomic promoter for expression of heterologous genes, is mutagenizedas described above. This process results in isolation of a population ofheterogeneous plasmid containing the random mutations. In parallel, thedesired heterologous cell surface protein or marker peptide gene iscloned into a shuttle vector for insertion into the mutagenized pTE3′2Jvector. Preferred cell surface proteins for use as markers include, butare not limited to, human B7.1 (Freeman et al., J. Immunol.143:2714-2722, 1989) and the murine H-2K^(b) class I molecule (Song etal., J. Biol. Chem. 269:7024-7029, 1994). The human B7.1 gene isamplified by standard three-cycle PCR, with 1.5 minute extension, from apCDM8 vector containing the full-length cDNA sequence (Freeman et al.,ibid), using the following oligonucleotide primers that are designed tocontain flanking Xba I and Bam HI sites.

Forward primer: hB7.1 FX (5′-rest. site/B7.1 sequence) (SEQ. ID. NO. 24)

5′-ATATATCTAGA/GCCATGGGCCACACACGGAGGCAG-3′

Reverse primer: hB7.1 RB (5′-rest. site/B7.1 sequence) (SEQ. ID. NO. 25)

5′-ATATAGGATCC/CTGTTATACAGGGCGTACACTTTC-3′

Following amplification, the approximately 875 bp DNA fragment ispurified using a QIAquick-spin PCR purification kit (Qiagen, Chatsworth,Calif.), digested with Xba I and Bam HI and ligated into Sindbis shuttlevector pH3′2J1 (Hahn et al., ibid) that also has been digested with XbaI and Bam HI and treated with calf intestinal alkaline phosphatase, tocreate the construct pH3′7.1.

Following random mutagenesis of the pTE3′2J double subgenomic vector, asdescribed above, plasmid pH3′—7.1 and the mutated population of plasmidpTE3′2J are digested with Apa I and Xho I, purified from 0.7% agarosegels using GENECLEAN II II™ (Bio101, Vista, Calif.), and ligated to forma heterogeneous population of a B7.1 expression vector, designatedpTE3′—7.1. Without transforming E. coli and isolating individual clones,the entire population of ligated vector is linearized with Xho I andused as template for in vitro SP6 transcription reactions, as describedabove. The heterogeneous population of randomly mutagenized B7.1 vectortranscripts is then electroporated into the desired cell type (forexample, BHK cells) for initiation of the Sindbis virus replicationcycle. Selection for virus mutants which establish a persistentinfection or exhibit reduced, delayed, or no inhibition of hostmacromolecular synthesis is performed using a monoclonal antibodyspecific for B7.1 (Pharmingen, San Diego, Calif.) and either magnetic-or fluorescence-activated cell sorting protocols. The preferredsecondary antibody tags include rat-anti-mouse IgG conjugated withmagnetic microbeads for magnetic cell sorting (miniMACS MagneticSeparation System, Miltenyi Biotec, Auburn, Calif.; Miltenyi et al.,Cytometry 11:231-238, 1990), and FITC-conjugated rat anti-mouse IgG(Pharmingen, San Diego, Calif.) for fluorescence activated cell sorting.Using such an approach and harvesting cells after an extended period(see above), only viable cells which contain a functional virus-derivedvector (as evidenced by B7.1 expression), displaying the desiredphenotype, are enriched.

Specifically, the heterogeneous population of randomly mutagenized B7.1vector transcripts is electroporated into 1×10⁷ cells, according to theprocedure of Liljestrom and Garoff (1991, ibid), and plated as a flatstock culture. At 12,24, 36, and 48 hour post-infection, the cellmonojayer is washed twice with fresh media to remove dead cells, andreplaced with media consisting of a mixture of 50% fresh media and 50%conditioned media. After a desired time post-infection (for example 72hours), the remaining cells are gently trypsinized to detach them fromthe culture dish, and pelleted by centrifugation at 1000 rpm, 4° C. Thecells are resuspended in 2 ml of blocking solution (PBS+10% fetal calfserum+1% BSA), incubated on ice for 10 minutes, and re-pelleted. Next,the cells are resuspended in 200 ul of the primary anti-B7.1 antibodysolution (diluted in PBS+0.5% BSA), and incubated on ice for 30 minutes.The cells are washed twice with PBS+0.5% BSA, pelleted, and resuspendedin 200 ul of magnetic bead solution (200 ul washed magnetic ratanti-mouse coated beads in PBS+0.5% BSA+5 mM EDTA). Following incubationat 4° C. for 30 minutes, the bead-bound cells are washed twice withPBS+0.5% BSA+5 mM EDTA, and resuspended in 1 ml of the same buffer. Thebead-bound cells are then purified using the MiniMacs magnet column,according to the manufacturer's directions. The eluted positive cellsare then re-seeded directly into a tissue culture dish containing asemi-confluent uninfected cell monolayer, at a ratio of 1 infected cellfor every 10⁴ uninfected cells. Additional rounds of selection areperformed as above for amplification/enrichment. The selected vectorvariants with the desired phenotype are isolated by limiting dilution orplaque purification, subjected to cDNA cloning, and the sequencesresponsible for the variant phenotype are isolated and characterized, asdescribed previously.

Alternatively, the B7.1 expression vector, pTE3′B7.1, may be useddirectly for in vitro transcription without prior mutagenesis. Followingtransfection of these RNA transcripts, selection for mutants of thedesired phenotype is performed as described above.

2. Vectors Expressing a Selectable Marker

Alternatively, an antibiotic resistance marker can be used for selectionof virus vector variants exhibiting the desired phenotype (FIG. 8F). Forexample, the Sindbis vector pRSIN-βgal (Dubensky et al., ibid) wasmodified by replacement of the βgalactosidase reporter gene with aneomycin phosphotransferase selectable marker and either subjected toprior mutagenesis or used directly. The gene encoding neomycin (G418)resistance was isolated by standard three-cycle PCR amplification, with1.5 minutes extension, from plasmid pcDNA3 (Invitrogen, San Diego,Calif.), using the following oligonucleotide primers that were designedto contain flanking Xho I and Not I restriction sites:

Forward primer: NeoFX (5′-rest. site/neo sequence) (SEQ. ID. NO. 26)

5′-ATATACTCGAG/ACCATGATTGAACAAGATGGATTG-3′

Reverse primer: NeoRN (5′-rest. site/neo sequence) (SEQ. ID. NO. 27)

5′-TATATAGCGGCCGC/TCAGAAGAACTCGTCAAGAAG-3′

Following amplification, the DNA fragment was purified withQIAquick-spin, digested with Xho I and Not I, and ligated into pRSIN-μalvector that also had been digested with Xho I and Not I, treated withcalf intestinal alkaline phosphatase, and purified from a 0.7% agarosegel, away from its previous βgalactosidase insert, using GENECLEAN II.The newly constructed Sindbis expression vector containing the neomycinresistance marker was designated pSin-Neo. Plasmid pSin-Neo waslinearized with Pme I, either directly or with 1,2, or 3 rounds of priormutagenesis by passage through E. coli strain XL-1 Red. The linear DNAwas used as template for in vitro SP6 transcription reactions and vectortranscripts were then transfected into the desired cell type (forexample, BHK cells) for initiation of the Sindbis replication cycle andheterologous gene expression. Approximately 24 hour post-transfection,the BHK cells were trypsinized and replated in media containing 0.5mg/ml G418. Subsequently, the media was changed at approximately 24 hourintervals to remove dead cells, and replaced with G418-containing mediaconsisting of a mixture of 50% fresh media and 50% conditioned media.Media changes were reduced after the majority of dead cells were washedaway, and cell foci began to form. At this time, all cells in controlplates transfected with Sindbis vector RNA expressing only a reportergene were killed by the drug. Using this selection, only viable cellswhich contained a functional Sindbis virus-derived vector, exhibitingthe desired phenotype (as evidenced by neomycin resistance), wereenriched. Stably transformed neomycin-resistant pools were obtainedusing this approach for both mutagenized and unmutagenized templates,and the pools were subsequently characterized. Similar selectionapproaches were demonstrated to work in cells that express higher levelsof interferon(s), for example L929 cells.

Mutant vector variants displaying the desired phenotype were isolated byharvesting RNA directly from the stably transformed cells using RNAzol B(Tel-Test, Friendswood, Tex.), followed by poly A selection. The RNAswere analyzed by northern blot, using a neomycin phosphotransferase geneprobe, to demonstrate the presence of both genomic and subgenomic viralvector RNA species (FIG. 8G). Lanes S1, S2, and S3 represent RNA fromthree independently derived G418-resistant pools. The BHK lanerepresents untransfected cellular RNA, while the Sin-Neo lane representsthe original in vitro transcribed RNA vector. Clearly, significantdifferences in the ratios of genomic to subgenomic RNA are observedamong the pools, suggesting their derivation from vectors containingdifferent causal mutations. The isolated RNA also was used to transferthe neomycin resistance phenotype to naive cells. Specifically, BHK-21cells were transfected with the above isolated RNA or a control templateRNA and treated with the G418 drug. Those cells transfected with theisolated RNA were immediately resistant to the drug and grew toconfluence within days, unlike the control transfections. Finally,complementation assays were performed using a defective Sindbis virus,galactosidase vector (designated Sin-d1-μgal), which is deleted ofnonstructural gene sequences between nucleotides 422 and 7054.Expression of the μ-galactosidase reporter from such a defective vectorcan occur only after the deleted nonstructural proteins are provided intrans. Transfection of Sin-dl-μgal RNA into the above G418-resistantpools resulted in expression levels of μ-galactosidase not seen insimilarly transfected control BHK cells.

To map the genetic locus of the Sindbis vectors responsible for reducedcytopathogenicity, PCR primer pairs were synthesized, allowing divisionand gene substitution of the vector sequences (excluding the 3′ -endUTR) in three distinct sections: nts. 1-2288, nts. 2289-4845, and nts.4846-7644 (FIG. 8H). These primer pairs may be used to amplify cDNAdirectly from vector variant RNA isolated from G418-resistant cells. Forexample, RNA from the S2 pool (see FIG. 8G) was subjected to cDNAcloning and substitution into a wild-type pSin-Neo vector. Following invitro transcription, wild-type and S2 mutant vector RNA was transfectedinto BHK cells and G418 drug selection was applied. Only the S2 mutantvector RNA resulted in rapid drug resistance and confluent cell growthwithin days, suggesting that the causal mutation resided within thisregion of gene replacement. Sequence analysis between the wild-type andS2 mutant vectors within this region, revealed a Proline to Threoninesubstitution at nsP2 amino acid 726, within the highly conservedalphavirus Leu-Xaa-Pro-Gly-Gly motiff. In addition, gene replacement ofthe same region with cDNA derived from pool S1 (see FIG. 8G) did notresult in a vector producing similar G418 resistant cells (FIG. 8H), nordid the substituted sequence contain a mutation within the conservedLeu-Xaa-Pro-Gly-Gly motiff. These data indicate that an alternativemutation outside the region of replacement is required for the observedphenotype.

Alphavirus Strain* Pro-Gly-Gly Region nsP2 a.a.'s (P-G-G) 1. Sindbisvirus Leu-Asn-Pro-Gly-Gly-Thr a.a. = 726-728 2. Sin-Neo S2 vectorLeu-Asn- Thr -Gly-Gly-Thr a.a. = 726-728 3. S.A.AR86 virusLeu-Asn-Pro-Gly-Gly-Thr a.a. = 726-728 4. Ockelbo virusLeu-Asn-Pro-Gly-Gly-Thr a.a. = 726-728 5. Aura virusLeu-Lys-Pro-Gly-Gly-Thr a.a. = 725-727 6. Semliki Forest virusLeu-Lys-Pro-Gly-Gly-Ile a.a. = 718-720 7. VEE virusLeu-Asn-Pro-Gly-Gly-Thr a.a. = 713-715 8. Ross River virusLeu-Xaa-Pro-Gly-Gly-Ser a.a. = 717-719

In another example, a Semliki Forest virus-derived vector, pSFV-1(GIBCO/BRL), was used for insertion of the antibiotic resistance markerand subsequent selection of the desired phenotype. The gene encodingneomycin (G418) resistance was isolated by standard three-cycle PCRamplification, with 1.5 minutes extension, from plasmid pcDNA3(Invitrogen, San Diego, Calif.), using the following oligonucleotideprimers that were designed to contain flanking BamH I restriction sites:

Forward primer: 5′BAMHI-Neo (SEQ. ID. NO. 118)

5′-ATATAGGATCCTTCGCATGATTGAACAAGATGGATTGC-3′

Reverse primer: 3′BAMHI-Neo (SEQ. ID. NO. 57)

5 ′-ATATAGGATCCTCAGAAGAACTCGTCAAGAAGGCGA-3′

Following amplification, the DNA fragment was purified withQIAquick-spin, digested with BamH I, and ligated into pSFV-1 vector thatalso had been digested with BamH I, treated with calf intestinalalkaline phosphatase, and purified from a 0.7% agarose gel, usingGENECLEAN II. The resulting SFV vector construct containing the neomycinresistance marker was designated SFV-Neo. In vitro transcription of RNAvector from mutagenized or unmutagenized SFV-Neo DNA template wasperformed and transfection, followed by selection for mutants of thedesired phenotype, was carried out essentially as described above forSindbis virus vectors. Several independently-derived, stably transformedG418-resistant pools were obtained and characterized. Northern blotanalysis for two such pools, SF1 and SF2, are shown in FIG. 8G, alongwith the original control RNA vector transcript (SFV-Neo). These datademonstrate that the selection methods described in the presentinvention have utility for multiple RNA virus vector systems.

Example 3 Preparation of SIN1-Based RNA Vector Replicons

A. Construction of the SIN-1 Basic Vector

SIN-1 derived vector backbones were constructed and inserted into aplasmid DNA containing a bacteriophage RNA polymerase promoter, suchthat transcription in vitro produced an RNA molecule that acts as aself-replicating molecule (replicon) upon introduction into susceptiblecells. The basic SIN-1 RNA vector replicon was comprised of thefollowing ordered elements: SIN-1 nsPs genes, subgenomic RNA promoterregion, a polylinker sequence, which may contain heterologous sequenceinsertions, the SIN-1 3′ non translated region (NTR), and a polyadenylate sequence. In addition, nsP genes of the desired phenotype,derived using methods such as those of Example 2, also may besubstituted. Following transfection into susceptible cells, autonomousreplication of the RNA vector replicon occurs as for virus, and theheterologous sequences are synthesized as highly abundant subgenomicmRNA molecules, which in turn serve as the translational template forthe heterologous gene product.

The 5′ region of the vector, comprised of the SIN-1 nsP genes andsubgenomic promoter, extends to within two nucleotides of the capsidgene translational initiation point. This region was first inserted intothe pKSII+plasmid (Stratagene) between the Apa I and Xho I sites. The 5′region of the vector was amplified by PCR from the pRSIN-1 g plasmid intwo overlapping fragments. The first fragment was generated in a PCRreaction with the following primer pair:

Forward primer: SP6-1F(Apa I site/SP6 promoter/SIN nts 1-18): (SEQ. IDNO. 12)

5′-TATATGGGCCCGATTTAGGTGACACTATAGATTGACGGCGTAGTACAC

Reverse primer: SIN5160R (SIN nts 5160-5140): (SEQ. ID NO. 28)

5′-CTGTAGATGGTGACGGTGTCG

The second fragment was generated in a PCR reaction with the followingprimer pair:

Forward primer: 5079F (SIN nts 5079-5100): (SEQ. ID NO. 29)

5′-GAAGTGCCAGAACAGCCTACCG

Reverse primer: SIN7643R (buffer sequence/Xho I site/SIN nts 7643-7621):(SEQ. ID NO. 30)

5′-TATATCTCGAGGGTGGTGTTGTAGTATTAGTCAG

The two PCR reactions were performed with the primer pairs shown aboveusing the Thermalase (Amresco, Solon, Ohio), Vent (New England Biolabs,Beverly, Mass.) or KlenTaq thermostable DNA polymerases. Additionally,the reactions contained 5% DMSO and “HOT START WAX” beads (Perkin-Elmer,Foster City, Calif.). The PCR amplification protocol shown below wasused. The extension period was 5 minutes or 2.5 minutes for reactionswith the SP6-1F/SIN5160R or 5079F/SIN7643R primer pairs, respectively.

Temperature (° C.) Time (Min) No. Cycles 95 2 1 95 0.5 55 0.5 35 72 5.0or 2.5 72 10 10

Following PCR, the two amplified products of 5142 bp (SP6-1F/SIN5160Rprimer pair) and 2532 bp (5079F/SIN7643R primer pair) were purified (PCRpurification kit, Qiagen, Chatsworth, Calif.), and digested with Apa Iand Sfi 1(5142 bp amplicon product) or Sfi I and Xho I (2532 bp ampliconproduct). The digested products were purified with GENECLEAN II (Bio101, Vista, Calif.) and ligated together with pKS+ plasmid (Stratagene,La Jolla, Calif.) prepared by digestion with Apa I and Xho I, andphosphatased with CIAP. This construction is known as pKSSIN-1-BV5′.

The 3′ region of the vector, comprised of the viral 3′ end, apolyadenylate tract, and a unique restriction recognition sequence wereinserted between the Not I and Sac I sites of the plasmid pKSSIN-1-BV5′.The 3′ region of the vector was amplified by PCR from the pRSIN-1 gplasmid in a reaction containing the following primer pair:

Forward primer: SIN11386F (buffer sequence/Not I site/SIN nts11386-11407): (SEQ. ID NO. 31)

5′ -TATATATATATGCGGCCGCCGCTACGCCCCAATGATCCGAC

Reverse primer: SIN11703R (buffer sequence/Sac I and Pme I sites/T40/SINnts 11703-11698): (SEQ. ID NO. 32)

5′ -CTATAGAGCT CGTTTAAACT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTTTTGAAATG

In addition to the primer pairs shown above, the PCR reaction containedThermalase, Vent or KlenTaq thermostable DNA polymerase, 5% DMSO, and“Hot Start Wax” beads (Perkin-Elmer). The amplification protocol was asshown above, but with a 72° C. extension period of 30 seconds.

The 377 bp amplified product corresponding to the 3′ vector end waspurified, digested with Not I and Sac I, purified, and ligated intopKSSIN-1-BV5′, which was prepared by digestion with Not I and Sac I andtreatment with CIAP. This plasmid is known as pKSSIN-1-BV.

Using techniques described above, the lacZ gene encoding theβ-galactosidase reporter protein was liberated from the plasmidpSV-β-galactosidase (Promega Corp., Madison, Wis.) with Bam HI and HindIII, and inserted into pKS+ at the corresponding enzyme recognitionsites. The lacZ gene was digested from this plasmid, pKS-β-gal, with XhoI and Not I, and inserted into pKSSIN-1-BV, between the Xho I and Not Isites. This plasmid is known as SINrep/SIN-1 nsP1-4/lacZ.

Alternatively, the firefly luciferase gene encoding the luciferasereporter protein was liberated from the plasmid pT3/T7-LUC (Clontech,Palo Alto, Calif.) by digestion with Hind III, and inserted into pKS+(Stratagene, La Jolla, Calif.) at the corresponding enzyme recognitionsites contained in the multiple cloning sequence to generate pKS-luc.The luciferase gene was liberated from pKS-luc by digestion with Xho Iand Not I and inserted into Xho I/Not I digested pKSSIN-1-BV. Thisplasmid is known as SINrep/SIN-1 nsP1-4/luc.

Additionally, the gene encoding the secreted form of alkalinephosphatase (SEAP) was inserted into pKSSIN-1-BV. Briefly, the SEAP genewas liberated from the plasmid pCMV/SEAP (Tropix, Bedford, Mass.) bydigestion with Hind III and Xba I, and inserted in pSK+ (Stratagene, LaJolla, Calif.) at the corresponding recognition sites contained in themultiple cloning sequence, to generate pSK-SEAP. The SEAP gene was thenliberated from pSK-SEAP by digestion with Xho I and Not I, and insertedinto the corresponding enzyme recognition sites of pKSSIN-1-BV. Thisplasmid is known as SINrep/SIN-1 nsP1-4/SEAP.

The individual SIN-1 nsP genes were substituted into the correspondingwild-type virus region of the Sindbis virus-based lac Z replicondescribed previously (Bredenbeek et al., J. Virol. 67:6439-6446, 1993),in order to compare the expression properties of the SIN-1 and wild-typeexpression vectors. Substitution of the SIN-1 nsP genes into theToto1101 -derived lac Z replicon was accomplished as described inExample 1. These vectors were designated as shown in the table below.

nsp Genes Origin Replicon Designation Toto1101 SIN-1 SINrep/lacZ nsP 1-4SINrep/SIN-1 nsP2/lacZ nsP 1,3-4 nsP 2 SINrep/SIN-1 nsP3/lacZ nsP 1-2, 4nsP 3 SINrep/SIN-1 nsP1-4/lacZ nsP 1-4

SP6 transcripts were prepared from the replicons shown in the tableabove, after linearization with Xho I, as described in Example 1. RNAtranscripts contained a 5′ sequence that is capable of initiatingtranscription of Sindbis virus, Sindbis virus nonstructural proteingenes 1-4, RNA sequences required for packaging, a Sindbis virusjunction region, the lacZ gene, and the Sindbis virus 3′ end proximalsequences required for synthesis of the minus strand RNA.

The in vitro transcription reaction products or purified RNA wereelectroporated into baby hamster kidney-21 (BHK-21) cells as describedpreviously (Liljestrom and Garoff, Bio/Technolog 9:1356-1361, 1991).Alternatively, BHK-21 cells were complexed with a commercial cationiclipid compound as described in Example 1 and applied to BHK-21 cellsmaintained at 75% confluency. Transfected cells were propagated in 35 mmdishes, and incubated at 37° C.

The efficiency of transfection of BHK-21 cells with SINrep/lac Z RNAsafter 9 hours was determined by two alternative methods. In the firstmethod, transfected cells expressing μ-galactosidase were determined bydirect staining with X-gal(5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside), after first fixingcells with 2% cold methanol, as described previously (MacGregor et al.,Cell Mol. Genet. 13:253-265, 1987). In the second method, expression ofμ-galactosidase were determined by immunofluorescence, using a rabbitanti-μ-galactosidase antibody. A portion of the cells transfected byeither method described above were propagated on circular glasscoverslips, contained in 35 mm dishes. At 9 hours post transfection(hpt), the media was removed by aspiration, and the cells were rinsedtwice with PBS, and fixed with methanol by incubation overnight at −20°C. The methanol was removed by aspiration, the cells were rinsed threetimes with PBS, then incubated with 2% BSA (fraction V, Sigma, St.Louis, Mo.) for 30 minutes at room temperature. Following incubationwith BSA to prevent non-specific antibody binding, the cells wereincubated with the primary anti-p-galactosidase antibody (diluted 1:800in 0.1% BSA/PBS) for 1 hour at room temperature. Excess primary antibodywas then removed by aspiration and rinsing three times with 0.1% BSA inPBS. Following 1:100 dilution in 2% BSA in PBS, 100 ml of goatanti-rabbit-FITC conjugate secondary antibody (Sigma, St. Louis, Mo.)were added to the coverslips and incubated for 45 minutes at roomtemperature, in the dark. Excess secondary antibody was removed byrinsing the coverslips twice with 0.1% BSA in PBS, and once with PBS.The coverslips were then mounted cell side down on a drop of Cytoseal 60mounting media (Stephens Scientific, Riverdale, N.J.), placed on amicroscope slide. Fluorescence microscopy was used to determine thefrequency of cells expressing μ-galactosidase, in order to determine thetransfection efficiency.

The level of μ-galactosidase in whole transfected cell lysates wasdetermined at 9 hpt, by two alternative methods. In the first method,transfected cells were rinsed with PBS after aspiration of the media,and 250 μl of reporter lysis buffer (Promega, Madison, Wis.) per 10⁶cells was added to each dish. μ-galactosidase expression levels weredetermined by mixing the supernatant fraction from cell lysates,processed by micro centrifugation at 14, 000 r.p.m. for 1 minute at roomtemperature, with a commercially available substrate detection system(Lumi-gal, Clontech, Palo Alto, Calif.), followed by luminometry(Analytical Luminescence Laboratory, San Diego, Calif.). In the secondmethod, the activity of μ-galactosidase was determined as describedpreviously by Sambrook and Maniatis (1989,2nd ed. Cold Spring HarborLaboratory Press, N.Y.). Briefly, transfected cells were rinsed with PBSafter aspiration of the media, and 200 μl of TTE lysis buffer (10 mMTris-HCl pH 8.0/1 mM EDTA/0.2% Triton X-100) per 10⁶ cells was added toeach dish. Following pelleting of cell debris from the cell lysate bymicro centrifugation at 14, 000 r.p.m. for 1 minute at room temperature,50 μl of the supernatant was added to 0.5 ml of Z buffer pH 7.0 (60 mMNa₂HPO₄/40 mM NaH₂PO₄/10 mM KCI/10 mM MgS04/50 mM 2-mercaptoethanol) andincubated at 37° C. for 5 minutes. μ-galactosidase activity in sampleswas then determined by spectrophotometry (420 nm) after addition of 0.2ml of a solution containing 4 mg/ml of the chromogenic substrateo-nitrophenyl-b-D-galactopyranoside (ONPG; SIGMA, St. Louis, Mo.), in Zbuffer. The samples were incubated at 37° C. until the yellow colordeveloped (approximately 5 minutes), and the reactions were terminatedby addition of 0.5 ml of 0.5 M Na₂CO₃.

The level of μ-galactosidase expression in transfected BHK-21 cells wasdetermined according to the methods described above, and is illustratedin the table shown below. The data indicated are normalized for varyingtransfection efficiency, as described above.

Expression of β-gal, Replicon Transfected relative to SINrep/lacZStandard Deviation SINrep/lacZ 1.0 SINrep/SIN-1 nsP2/lacZ 3.2 ±0.4SINrep/SIN-1 nsP3/lacZ 0.2 ±0.0 SINrep/SIN-1 nsP1-4/lacZ 5.2 ±1.3

The results demonstrate that the replicon vectors derived from the SIN-1variant strain are indeed functional. When transfected into BHK-21cells, the level of expressed reporter protein are higher than thatobserved in wild type virus-derived vector transfected cells.Furthermore, as with the phenotype for establishment of productivepersistent infection, the higher level of μ-galactosidase expression inBHK-21 cells transfected with the SIN-1-derived replicon vectors mappedprimarily to the nsP 2 gene.

Example 4 Preparation of Sin1-Based DNA Vectors

A. Construction of Plasmid DNA SIN-1 Derived Expression Vectors

Efficient initiation of the Sindbis virus infectious cycle can occur invivo from a genomic cDNA clone contained within an RNA polymerase IIexpression cassette (Dubensky et al., J. Virol 70:508-519, 1996. ). Theability to express functional alphavirus genes from a DNA format hasenabled two new alphavirus-based gene expression systems to bedeveloped: (1) a plasmid DNA-based vector with applications for geneticimmunization, and (2) the production of packaged alphavirus particles incells co-transfected with vector replicon and DH plasmid DNAs.Previously, molecular approaches to produce infectious Sindbis virus RNAand its derived complementary vectors were restricted primarily to invitro transcription of cDNA clones from a bacteriophage RNA polymerasepromoter followed by transfection into permissive cells.

The plasmid DNA-based alphavirus derived expression vector is known asELVS™ (Eukaryotic Layered Vector System). The ELVS™ plasmid DNA vectorinvolves the conversion of a self-replicating vector RNA (replicon) intoa layered DNA-based expression system. Within certain embodiments thefirst layer has a eukaryotic (e.g. RNA polymerase II) expressioncassette that initiates transcription of a second layer, whichcorresponds to the RNA vector replicon. Following transport of thereplicon expressed from the first layer from the nucleus to thecytoplasm, autocatalytic amplification of the vector proceeds accordingto the viral (e.g. alphavirus) replication cycle, resulting inexpression of the heterologous gene.

Construction of plasmid DNA expression vectors derived from SIN-1 virusor those variants selected as taught in Example 2 were performed withmodifications of methods previously described (Dubensky et al., J. Virol70:508-519, 1996). The expression vector was assembled on the plasmidvector pBGS 131 (ATCC No. 37443), which is a knamycin resistant analogueof pUC 9 (Spratt et al., Gene 41:337-342, 1986). pBGS131 and its derivedplasmids were propagated in LB medium containing 20 μg/ml kanamycin.

To facilitate insertion of heterologous sequences into the expressionvector, the Xho I recognition sequence, located within the translationalreading frame of the kanamycin gene in pBGS131, was removed by insertinga partially complementary 12-mer oligonucleotide pair that contained XhoI sticky ends. The Xho I recognition site was lost as a result of theinsertion, as shown below:

Oligonucleotide 1:(SEQ. ID NO.33)

5′-TCGATCCTAGGA

Original pBGS131 Paired pBGS131 sequence after sequence Oligonucleotidesinsertion of 12-mer SerArg SerIleLeuGlySerArg CTCGAGGC TCGATCCTAGGACTCGATCCTAGGATCGAGGC GAGCTCCG AGGATCCTAGT GAGCTAGGATCCTAGCTCCG

(Xho I site: CTCGAG) (SEQ. ID NOS. 33-36 and 104).

The oligonucleotide is gel annealed in equal molar concentrations in thepresence of 10 mM MgCl₂, heated to 100° C. for 5 min, cooled slowly toroom temperature, and phosphorylated with polynucleotide kinase. Theoligonucleotide was ligated at a 200:1 ratio of insert:plasmid vector topBGS131, which was prepared by Xho I digestion and CIAP treatment. Theresulting plasmid is called pBGS131 dlXho I. The growth rates ofXL1-Blue (Stratagene) transformed with pBGS131 or pBGS131dlXho Iplasmids in LB medium containing 20 μg/ml kanamycin wasindistinguishable over a time course between 1.5 and 8 hours.

The bovine growth hormone (BGH) transcriptiontermination/polyadenylation signal was inserted between the Sac I andEco RI sites of pBGS131 d1Xho I. The BGH transcription terminationsequences were isolated by PCR amplification using the primer pair shownbelow and the pCDNA3 plasmid (Invitrogen, San Diego, Calif.) astemplate.

Forward primerBGHTTF (buffer sequence/Sac I site/CDNA3 nts 1132-1161):(SEQ. ID NO. 37)

5′-TATATATGAGCTCTAATAAAATGAGGAAATTGCATCGCATTGTC

Reverse primer BGHTTR (buffer sequence/Eco RI site/pCDNA3 nts1180-1154): (SEQ. ID NO.38)

5′-TATATGAATTCATAGAATGACACCTACTCAGACAATGCGATGC

The primers shown above were used in a PCR reaction with a threetemperature cycling program, using a 30 sec extension period. The 58 bpamplified product was purified with the PCR purification kit (QiagenChatsworth. Calif.), digested with Sac I and Eco RI, purified withGENECLEAN II, and ligated into Sac I/Eco RI digested, CIAP treated pBGS131. The plasmid is known as pBGS 131 d1Xho I-BGHTT.

The 3′ end of the Sindbis virus-derived plasmid DNA expression vectorwas then inserted into the pBGS131 d1Xho I-BGHTT construct. This regionof the vector contains the following ordered elements: Sindbis virus 3′end non-translated region (3′ NTR); a 40-mer poly(A) sequence; thehepatitis delta virus (HDV) antigenomic ribozyme; and a Sac Irecognition sequence. Construction of these ordered elements wasaccomplished by nested PCR, using the primers shown below and thepKSSIN-1-BV plasmid (Example 4) as template.

Forward primer: SIN11386F (buffer sequence/Not I site/SIN nts11386-11407): (SEQ. ID NO. 31)

5′-TATATATATATGCGGCCGCCGCTACGCCCCAATGATCCGAC

Nested primer: DAHDV1F (poly(A)/HDV RBZ nts 1-46): (SEQ. ID NO. 39)

5′-AAAAAA GGGTCGGCAT GGCATCTCCA CCTCCTCGCG GTCCGACCTG GGCATC

Reverse primer: SacHDV77R (buffer sequence/Sac I site/HDV RBZ nts77-27): (SEQ. ID NO. 40)

5′-TATATGAGCTCCTCCCTTAGCCATCCGAGTGGACGTGCGTCCTCCTTCGGATGCCCAGGTCGGACCGCG

The primers shown above were used in a PCR amplification according tothe reaction conditions and three temperature cycling program describedin Example 4, with an extension time of 30 sec. The 422 bp amplifiedproduct was purified with a PCR purification kit (Qiagen Chatsworth,Calif.), digested with Not I and Sac I, purified with GENECLEAN II, andligated into Vot I/Sac I digested, CIAP-treated pKSSIN-1-BV. Thisconstruct is known as pKSSIN-1BV/HDVRBZ and contains Sindbisvirus-derived plasmid DNA expression vector sequences from the Bgl IIsite at Sindbis nt 2289 extending through the 3′ end of the vectorincluding the HDV ribozyme sequence.

Plasmid pKSSIN-1BV/HDVRBZ was then digested with Bgl II and Sac I, the5815 bp fragment was isolated by 1% agarose/TBE gel electrophoresis,purified with GENECLEAN II, and was inserted into Bgl II/Sac I digested,CIAP-treated pBGS131 d1Xho I-BGHTT to generate the plasmid constructknown as pBGfSIN-1Bg1LF. This construct contains the region of theSindbis virus expression vector from plasmid pKSSIN-1BV/HDVRBZ describedabove with the 3′ end fused to the BGH transcription terminationsequence on the pBGS 131 d1Xho I plasmid.

Assembly of the Sindbis virus plasmid DNA vector was completed byinsertion of the CMV promoter juxtaposed with the first 2289 nts of theSindbis virus genome (includes the 5′ viral end and a portion of thensPs genes) into the pBG/ SIN-1BglLF plasmid. Using an overlapping PCRapproach, the CMV promoter was positioned at the 5′ viral end such thattranscription initiation results in the addition of a single non-viralnucleotide at the 5′ end of the Sindbis virus vector replicon RNA. TheCMV promoter was amplified in a first PCR reaction from pCDNA3(Invitrogen, San Diego, Calif.) using the following primer pair:

Forward primer: pCBgl233F (buffer sequence/Bgl II recognitionsequence/CMV promoter nts 1-22): (SEQ. ID NO. 41)

5′-TATATATAGATCTTTGACATTGATTATTGACTAG

Reverse primer: SNCMV1142R (SIN nts 8-1/CMV pro nts 1142-1108): (SEQ. IDNO. 42)

5′-CCGTCAATACGGTTCACTAAACGAGCTCTGCTTATATAGACC

The primers shown above were used in a PCR reaction according to thereaction conditions and three temperature cycling program described inExample 4, with an extension time of 1 min.

The SIN-1 5′ end was amplified in a second PCR reaction from pKSRSIN-1 gclone (Example 1) using the following primer pair:

Forward primer: CMVSIN1F (CMV pro nts 1124-1142/SIN nts 1-20): (SEQ. IDNO. 43)

5′-GCTCGTTTAGTGAACCGTATTGACGGCGTAGTACACAC

Reverse primer: SIN 3182R (SIN nts 3182-3160): (SEQ. ID NO. 44)

5′-CTGGCAACCGGTAAGTACGATAC

The primers shown above were used in a PCR reaction with a threetemperature cycling program using a 3 min extension period.

The 930 bp and 3200 bp amplified products were purified with a PCRpurification kit (Qiagen) and used together in a PCR reaction with thefollowing primer pair:

Forward primer: pCBg233F: (SEQ. ID NO. 41)

5′-TATATATAGATCTTTGACATTGATTATTGACTAG

Reverse primer: (SIN nts 2300-2278): (SEQ. ID NO. 45)

5′-GGTAACAAGATCTCGTGCCGTG

The primers shown above were used in a PCR reaction with a threetemperature cycling program using a 3.5 min extension period.

The 26 3′ terminal bases of the first PCR amplified product overlap withthe 26 5′ terminal bases of the second PCR amplified product; theresultant 3200 bp overlapping secondary PCR amplified product waspurified by 1% agarose/TBE electrophoresis, digested with Bgl II, andligated into Bgl II digested, CIAP-treated pBG/SIN-1Bg1LF. Thisconstruct is called pBG/SIN-1 ELVS 1.5.

As discussed within Example 1, relatively few nucleotide point changesin the nsP gene sequence of wild-type Sindbis virus result in thephenotype characteristic of SIN-1. No new restriction enzyme recognitionsites are generated as a result of these nucleotide changes whichfacilitate clones derived from wild-type and SIN-1 genotypes to beeasily distinguished. A PCR-based diagnostic assay was therefore devisedas a rapid method for identification of SIN-1 derived clones. Briefly,forward primers were designed so that a particular base change betweenSIN-1 and wild-type was positioned at the 3′ terminal base of theprimer. One primer contained the SIN-1 nucleotide while anothercontained the wild-type nucleotide. A reverse primer in a regiondownstream conserved between both genotypes was used in combination witheach forward primer. At the correct annealing temperature, SIN-1templates were only amplified in reactions containing SIN-1 forwardprimers. The primer sequences used to distinguish wild-type and SIN-1genotypes is given below. The reaction conditions were as describedthroughout the examples contained herein.

Primer Set I:

Forward primers:

WT100F 5′-GTC CGT TTG TCG TGC AAC TGC (SEQ. ID NO. 105)

SIN-1100F: 5′-GTC CGT TTG TCG TGC AAC TGA (SEQ. ID NO. 106)

Reverse primer:

SIN2300R

PCR Program: (95° C.-30″, 72° C.-2′) 20 cycles

Primer Set 2:

Forward primers:

WT3524F 5′-CAA TCT TCC TCA CGC CTT AGC (SEQ. ID NO. 107)

SIN-13524F 5′-CAA TCT TCC TCA CGC CTT AGT (SEQ. ID NO. 108)

Reverse primer:

SIN5448R

PCR Program: (95° C.-30″, 60° C.-30″, 72° C.-2′) 20 cycles

Primer Set 3:

Forward primers:

WT7592F 5′TCC TAA ATA GTC AGC ATA GTA (SEQ. ID NO. 109)

SIN-17592F 5′TCC TAA ATA GTC AGC ATA GTT (SEQ. ID NO. 110)

Reverse primer:

SIN7643R 5′-TATATCTCGAGGGTGGTGTTGTAGTATTAGTCAG (SEQ. ID NO. 111)

PCR Program: (95° C.-30″, 60° C.-30″, 72° C.-2′) 20 cycles

Reporter protein expression vectors were constructed by inserting thelacZ, SEAP, or luciferase reporter genes into the pBG/SIN-1 ELVS 1.5vector backbone. In separate reactions, the pKS-β-gal, pSK-SEAP, andpKS-luc plasmids (Example 4), were digested with Xho I and Not I. Thefragments containing the lacZ, SEAP, or luciferase genes were isolatedby 1% agarose/TBE gel electrophoresis and purified subsequently withGENECLEAN II. These reporter genes were then ligated in separatereactions with Xho I/Not I digested, CIAP-treated pBG/SIN-1 ELVS 1.5plasmid. These constructs are known as pBG/SIN-1 ELVS 1.5-β-gal,pBG/SIN-1 ELVS 1.5-SEAP, and pBG/SIN-1 ELVS 1.5-luc.

B. Expression of Heterologous Proteins in Cells Transfected withpBG/SIN-1 ELVS 1.5-SEAP, pBG/SIN-1 ELVS 1.5-luc or pBG/SIN-1 ELVS1.5-β-gal Expression Vectors

The pattern of secreted alkaline phosphatase, luciferase, andβ-galactosidase reporter gene expression in BHK cells transfected withpBG/SIN-1 ELVS 1.5 or pBG/wt ELVS 1.5 vectors was compared. The pBG/wtELVS 1.5 plasmid contains sequences derived from wild-type Sindbisvirus, rather than the SIN-1 variant. Construction of the pBG/wt ELVS1.5 expression vectors was exactly as described herein for the pBG/SIN-1ELVS 1.5 expression vectors, except that full-length genomic cDNAderived from wild-type Sindbis virus (Dubensky et al., WO 95/07994) wasused as the template for the vector construction. Construction of thepBG/wt ELVS 1.5 expression vector has been described previously(Dubensky, supra.); thus, although the strains, and therefore thesequences, are different, the Sindbis virus-specific regions containedin the pBG/wt ELVS 1.5 and pBG/SIN-1 ELVS 1.5 expression vectors are thesame.

Baby hamster kidney-21 (BHK-21) cells maintained at 75% confluency in 12mm dishes were transfected with 1.0 Mg of pBG/SIN-1 ELVS 1.5 or pBG/wtELVS 1.5 expression vector plasmid DNAs complexed with 4.0 μl of acommercially available lipid (Lipofectamine, GIBCO-BRL). Otherwise,transfection conditions were as suggested by the lipid manufacturer.Eagle minimal essential medium supplemented with 5% fetal bovine serawas added to the cells at 4 hours post transfection (hpt), unlessotherwise indicated. Transfected cells were incubated at 37° C. Atvarious times post transfection, as indicated below, several assays wereperformed to compare vector-specific RNA synthesis, and expression ofsecreted alkaline phosphatase, luciferase, or β-galactosidase reportergene expression in cells transfected with pBG/SIN-1 ELVS 1.5 or pBG/wtELVS 1.5 plasmid DNAs.

The levels of alkaline phosphatase secreted into the culture medium ofBHK cells transfected with pBG/SIN-1 ELVS-1 1.5-SEAP or pBG/wt ELVS1.5-SEAP plasmid DNA were compared. Cell culture medium was assayed forthe presence of alkaline phosphatase with the Phospha-Light⊥chemiluminescent reporter gene assay, according to the directions of themanufacturer (Tropix, Inc., Bedford, Mass.). Briefly, 10 μl of cellculture supernatant was mixed with 30 μl of Dilution Buffer andincubated for 30 minutes at 65° C. The sample was allowed to cool toroom temperature before mixing with 40 μl of Assay Buffer. The samplewas incubated for five minutes at room temperature followed by theaddition of 40 μl of Reaction Buffer. Samples were incubated for 20minutes at room temperature. Total luminescence was measured on anML3000 microtiter plate luminometer (Dynatech, Inc., Chantilly, Va.) incycle mode. and alkaline phosphatase (AP) in the culture medium of BHKcells transfected with pBG/SIN-l ELVS-1 1.5-SEAP or pBG/wt ELVS 1.5-SEAPplasmid DNAs, were determined at 48 hpt, and the results are shown inthe table below.

Plasmid Transfected RLU at 48 hpt pBG/SIN-1 ELVS 1.5-SEAP   18 ± 1.7pBG/wt ELVS 1.5-SEAP   94 ± 10.7 pCDNA3 0.13 ± 0.04

Additionally, the levels of vector-specific RNAs synthesized in BHK-21cells transfected with pBG/SIN-1 ELVS 1.5-SEAP or pBG/wt ELVS 1.5-SEAPplasmids were determined by Northern blot analysis, exactly as describedpreviously (Dubensky, supra.), at 48 hours post-transfection. Theresults of this experiment are shown in FIG. 9A. Total cellular RNA wasisolated from transfected BHK cells with Tri-Reagent as described by themanufacturer (Molecular Research Center. Inc., Cincinnati, Ohio). Totalcellular RNA concentrations present in samples from transfected BHKcells were determined spectrophotometrically. Additionally, materialisolated from transfected cells was determined to be intact byelectrophoresis of 0.5 ug of total cellular RNA through 0.7% agarose/TBEmini gels, stained 10 μl/ml of ethidium bromide. Northern blot analysiswas performed according to Sambrook and Maniatis (1989, 2nd ed. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.). In order that RNAfrom all transfected samples could be visualized on a autoradiogram froma single Northern blot analysis, 2.5 ug and 30 ug of RNA were loaded perlane from pBG/wt ELVS 1.5-SEAP and pBG/SIN-1 ELVS 1.5-SEAP transfectedcells, respectively. Four samples of RNA, from individual transfectionswith both plasmids tested, were electrophoresed through 0.7%formaldehyde agarose gels and transferred to Zeta-probe membrane(Bio-Rad, Richmond, Calif.). The blot was hybridized with random-primedprobes corresponding to the alkaline phosphatase gene. The results ofthis experiment in which the levels of vector-specific RNA synthesis andAP expression in transfected BHK cells were compared at 48 hpt,demonstrate that while the level of vector-specific RNA synthesized incells transfected with pBG/SIN-1 ELVS 1.5-SEAP DNA was at least 100-foldlower than in pBG/wt ELVS 1.5-SEAP transfected cells, the levels of APwere only 5-fold lower in cells transfected with pBG/SIN-1 ELVS 1.5-SEAPDNA, compared to pBG/wt-ELVS 1.5-SEAP DNA.

The levels of alkaline phosphatase secreted into the culture medium ofBHK cells transfected with pBG/SIN-1 ELVS-1 1.5-SEAP or pBG/wt ELVS1.5-SEAP plasmid DNA were also compared over a 7 day time-course. Theresults of this study, illustrated in FIG. 9B, demonstrate that thelevels of AP present in the culture medium at early time points weremuch lower in cells transfected with pBG/SIN-1 ELVS-1 1.5-SEAP plasmid,compared to pBG/wt ELVS 1.5-SEAP plasmid. However, the level of APexpressed in cells transfected with the SIN-1 Sindbis virus variantstrain-derived vectors rapidly increased and was higher than in cellstransfected with wild-type virus-derived vectors by the 96 hpt timepoint.

The luciferase levels present in BHK cells transfected with pBG/SIN-1ELVS-1 1.5-luc or pBG/wt ELVS 1.5-luc plasmid DNA were compared at 24,48, and 72 hpt. The luciferase expression levels were quantitated byadding 250 μl of reporter lysis buffer (Promega, Madison Wis.) per 10⁶transfected cells. centrifuging the lysate at 14, 000 rpm for 1 min, andthen mixing the supernatant fraction from the cell lysates with acommercially available substrate detection system (Promega, MadisonWis.), followed by luminometry (Analytical Luminescence Laboratory, SanDiego, Calif.). The results from this experiment (shown in the tablebelow), and shown graphically in FIG. 10, parallel the results observedwith the alkaline phosphatase expression vectors. At early times posttransfection the luciferase expression levels were lower in BHK cellstransfected with pBG/SIN-1 ELVS 1.5-luc plasmid, compared to pBG/wt ELVS1.5-luc plasmid. However, at the 48 and 72 hpt time points, theluciferase levels were similar in BHK cells transfected with Sindbisvirus SIN-1 variant strain- and wild-type-derived expression vectors.

Hr. Post Trans- Relative Light Units Plasmid Transfected fection (Ave, ±SD) pBG/SIN-1 ELVS 1.5-luc 24 1.2 × 10⁹ pBG/wt ELVS 1.5-luc 2.1 × 10⁸pBG/SIN-1 ELVS 1.5-luc 48 3.3 × 10⁹ pBG/wt ELVS 1.5-luc 4.1 × 10⁹pBG/SIN-1 ELVS 1.5-luc 72 1.8 × 10⁹ pBG/wt ELVS 1.5-luc 5.8 × 10⁹ pCDNA348 482

The efficiency of transfection of the pBG/SIN-1 ELVS 1.5-β-gal andpBG/wt ELVS 1.5-β-gal plasmids in BHK cells at 48 hpt was determined bydirect X-gal (5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside) stainingof the cell monolayer (MacGregor, Cell Mol. Genet. 13:253-265, 1987), inorder to measure directly the number of cells expressingβ-galactosidase. The transfection efficiencies were equivalent, and areshown in the table below.

Plasmid Transfected No. Blue Cells/100X Field pBG/SIN-1 ELVS 1.5-β-gal24 ± 3 pBG/wt ELVS 1.5-β-gal 26 ± 7

The levels of vector-specific RNAs synthesized in BHK-21 cellstransfected with pBG/SIN-1 ELVS 1.5-β-gal or pBG/wt ELVS 1.5-β-galplasmids were determined by Northern blot analysis, exactly as describedpreviously (Dubensky, supra.), at 48 and 72 hours post-transfection.Total cellular RNA was isolated from transfected BHK cells withTri-Reagent as described by the manufacturer (Molecular Research Center,Inc., Cincinnati, Ohio). Total cellular RNA concentrations present insamples from BHK cells transfected with pBG/SIN-1 ELVS 1.5-β-gal orpBG/wt ELVS 1.5-β-gal plasmids were determined spectrophotometrically.Additionally, material isolated from transfected cells was determined tobe intact by electrophoresis of 0.5 ug of total cellular RNA through0.7% agarose/TBE mini gels, stained 10 ul/ml of ethidium bromide.Northern blot analysis was performed according to Sambrook and Maniatis(1989,2nd ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).In order that RNA from all transfected samples could be visualized on aautoradiogram from a single Northern blot analysis 2.5 ug and 5 ug ofRNA were loaded per lane from pBG/wt ELVS 1.5-β-gal and pBG/SIN-1 ELVS1.5-β-gal transfected cells, respectively. Two samples of RNA, fromindividual transfections with both plasmids tested, at 48 and 72 hpt,were electrophoresed through 0.7% formaldehyde agarose gels andtransferred to Zeta-probe membrane (Bio-Rad, Richmond, Calif.). The blotwas hybridized with random-primed probes corresponding to theβ-galactosidase gene. The results of this experiment, shown in FIG. 11A,demonstrate that the level of vector specific RNA synthesized in cellstransfected with pBG/SIN-1 ELVS 1.5-β-gal DNA at 48 and 72 hours posttransfection was at least 100-fold lower than the level of RNA detectedin pBG/wt ELVS 1.5-β-gal transfected cells.

Additionally the β-galactosidase expression levels were quantitated intransfected whole cell lysates by adding 250 μl of reporter lysis buffer(Promega, Madison Wis.) per 10⁶ transfected cells, centrifuging thelysate at 14, 000 rpm for 1 min, and then mixing the supernatantfraction from the cell lysates with a commercially available substratedetection system (Clontech, Palo Alto, Calif.), followed by luminometry(Analytical Luminescence Laboratory, San Diego, Calif.). The resultsfrom this experiment (shown in the table below, and graphically in FIG.9C) demonstrate that at early times post transfection theβ-galactosidase expression levels were significantly lower in BHK cellstransfected with pBG/SIN-1 ELVS 1.5-β-gal plasmid., compared to pBG/wtELVS 1.5-β-gal plasmid. However, reporter expression rapidly increasedover the time-course in pBG/SIN-1 ELVS 1.5-β-gal transfected cells suchthat the β-galactosidase levels were higher than in wild-type virustransfected cells at the final 120 hr time point.

Hr. Post Trans- Relative Light Units Plasmid Transfected fection (Ave, ±SD) pBG/SIN-1 ELVS 1.5-β-gal 24  54030 ± 7348 pBG/wt ELVS 1.5-β-gal4801590 ± 74,425 pBG/SIN-1 ELVS 1.5-β-gal 48  310830 ± 31083 pBG/wt ELVS1.5-β-gal 2214921 ± 248071 pBG/SIN-1 ELVS 1.5-β-gal 72 1443474 ± 98156pBG/wt ELVS 1.5-β-gal 3793524 ± 857336 pBG/SIN-1 ELVS 1.5-β-gal 962232585 ± 299166 pBG/wt ELVS 1.5-β-gal 3514262 ± 548225 pBG/SIN-1 ELVS1.5-β-gal 120 3200910 ± 128036 pBG/wt ELVS 1.5-β-gal 1986537 ± 166869pCDNA3   3637

Expression of 62 -galactosidase in cells transfected with the pBG/SIN-1ELVS-1 1.5-β-gal or pBG/wt ELVS 1.5-β-gal plasmid DNAs was also measureddirectly by Western blot analysis using a monoclonal antibody specificfor the reporter protein (Boehringer Mannheim), at the final 120 hpttime point. In parallel with the reporter protein activity determined at120 hpt, the level of β-galactosidase protein was at the same level, orgreater, in PBG/SIN-1 ELVS-1 1.5-β-gal transfected BHK cells, comparedto pBG/wt ELVS 1.5-β-gal, and is demonstrated in FIG. 11C.

Taken together, the results described herein demonstrate that the levelof vector-specific RNA synthesized is at least 100-fold less inpBG/SIN-1 ELVS transfected cells, compared to pBG/wt ELVS transfectedcells. Importantly however, after a 48-72 hour lag, the levels ofreporter gene expression are equivalent, or higher, in pBG/SIN-1 ELVStransfected cells, compared to pBG/wt ELVS transfected cells. Thephenotype of pBG/SIN-1 ELVS, characterized by high expression levelscombined with low vector-specific RNA synthesis in transfected cells, isdue likely to the diminished, or absent, inhibition of host cell proteinsynthesis. This property of pBG/SIN-1 ELVS thus results in much higherlevels of expressed reporter protein per subgenomic MRNA translationtemplate in transfected cells, compared to pBG/wt ELVS. In summary, thephenotype of the plasmid DNA expression vectors derived from the SIN-1variant strains follows the parent virus, in terms of equivalentexpression levels, combined with relatively low levels of RNA synthesis,compared to wild-type virus derived-vectors. As vectors do not containany of the Sindbis virus structural proteins, this phenotype must map tothe nonstructural genes of the SIN-1 virus variant.

Example 5 Modifications of Plasmid DNA SIN-1 Derived Expression Vectors

Expression levels of heterologous genes in target cells fromalphavirus-based vectors are affected by several factors, including hostgenus and vector configuration. For example, β-galactosidase expressionlevels are 10- to 100-fold higher in BHK cells, compared to some humancells, such as HT1080, transfected with pBG/ELVS vectors. The levels ofreporter gene expression in BHK and several human cell lines transfectedwith pBG/wt ELVS 1.5-βgal plasmid DNA (see example 4) were compared inorder to establish the relative level of vector-specific expression incell types derived from the intended in vivo target genus. The levels ofβ-galactosidase expression in BHK cells and HT1080 (ATCC CCL 121) cells,a human fibrosarcoma line, transfected with pBG/wt ELVS 1.5-βgalplasmid, or with a conventional plasmid expression vector, weredetermined. The conventional plasmid vector was constructed by insertionof the lac Z gene (Promega, Madison, Wis.) into the CMV promoter-drivenpUC-derived expression plasmid multiple cloning site (Invitrogen, SanDiego, Calif.), and is known as pCMV-β-gal. The results of this study,given in FIG. 12A, demonstrate that the level of β-galactosidaseexpression was nearly 100-fold lower in HT1080 cells transfected withpBG/wt ELVS 1.5-βgal plasmid DNA, compared to BHK cells. Cells were alsotransfected with pCMV-β-gal in order to segregate RNA polymerase IIexpression from Sindbis virus vector replicon expression. In thisexperiment, while the expression decreased 5- to 10-fold in HT1080 cellstransfected with pCMV-β-gal plasmid, compared to BHK cells, expressiondecreased nearly 100-fold in HT 1080 cells transfected with pBG/wt ELVS1.5-βgal plasmid DNA. Thus, the results indicate that the dramaticdecrease of reporter gene expression in HT1080 cells transfected withpBG/wt ELVS 1.5-βgal plasmid DNA is due in part to the diminishedactivity of the Sindbis virus vector replicon in these human cells.

Given the overall plasticity of the RNA alphaviral genome and thepropagation of virus in BHK cells. It is not surprising that theexpression levels of heterologous genes are highest in the host celllines from which the vectors were derived. Thus, selection ofalphaviruses with the SIN-1 phenotype (as described in Examples 1 and2), characterized by comparatively low viral RNA levels and equivalentvirus production levels, combined with delayed or absent inhibition ofhost cell protein synthesis, can be performed in any human primary, ordiploid or polyploid human cells.

In addition to selecting alphaviruses with desired phenotypes in cells(e.g., human) which more closely parallel target cells in vivo, severalalternative modifications of the prototype plasmid DNA expressioncassette components can also be performed. For example, substitution ofthe MoMLV RNA polymerase II promoter with the stronger CMV immediateearly (IE) promoter significantly enhances the level of heterologousgene expression in transfected cells (Dubensky et al., J. Virol.70:508-519, 1996, and Dubensky et al., W/O 95/07994). Further,juxtaposition of introns, for example SV40 small t antigen or CMV intronA, either upstream or downstream from the heterologous gene, canincrease the level of heterologous gene expression in some transfectedcell types (Dubensky et al., supra.).

Several further alternative modifications of the prototype plasmid DNAexpression cassette components can also be utilized in order to enhancethe overall expression in transfected cells in vitro or in vivo. In onemodification, the Hepatitis B virus (HBV) posttranscriptional regulatoryelement (PRE) was inserted in the pBG/wt ELVS 1.5-βgal plasmid DNA. ThePRE sequence activates the transport of HBV S transcripts in cis fromthe nucleus to the cytoplasm. The PRE sequence appears to functionindependently of splice donor and acceptor sites, and has been shown toactivate cytoplasmic expression of a β-globin transcript not containingintrons. It has been proposed that the PRE functions in cis to allow theexport of nuclear transcripts that do not interact efficiently with thesplicing pathway and hence are not exported well from the nucleus (Huanget al., Molecular and Cellular Biology 15:3864-3869, 1995).

The PRE sequence was cloned into pBG/SIN-1 ELVS 1.5-βgal by isolatingfirst a PCR-generated 564 bp fragment of HBV from the fill lengthgenomic clone of the ADW viral strain, pAM6 (ATCC No. 39630). Theamplified fragment extends from base 1238-1802 of the HBV genome. Theprimer sequences are given below.

Forward Primer: NTPRE1238F (SEQ. ID NO. 46)

5′-CCTATGCGGCCGCGTGGAACCTTTGTGGCTCCTC

Reverse Primer: EAPRE1802R (SEQ. ID NO. 47)

5′-CCTATTGGCCAGCAGACCAATTTATGCCTAC

The primers introduce a Not I recognition site at the 5′ end of thefragment and an Eae I recognition site at the 3′ end. Not I and Eae Ihave compatible sticky ends. The Eae I recognition site is internal tothe Not I site, so Eae I cuts both sites.

The PCR fragment was digested with Eae I and cloned into Not Idigested/CIAP treated pBG/wt ELVS 1.5-βgal. The correct clone retainsthe Not I site at the 5′ terminus of the PRE and is called pBG/wtELVS/PRE 1.5-βgal.

The possible effect of the PRE sequence contained in ELVS plasmids onheterologous gene expression in transfected cells was determined.Briefly, BHK and HT1080 cells were cultured in 12 mm dishes to 75%confluency and transfected with 500 ng of pCMV-β-gal, pBG/wt ELVS1.5-βgal, or pBG/wt ELVS/PRE 1.5-βgal plasmid DNA complexed withLipofectamine (GIBCO-BRL, Gaithersburg, Md.), and the level ofβ-galalactosidase expression was determined 48 hr later. Transfectionefficiencies were determined by direct Xgal staining of transfectedmonolayers, as described in Example 4, and are shown in the table below.

No. Blue cells/12 mm Dish Construct BHK HT1080 pCMV-β-gal 549 42 pBG/wtELVS 1.5-βgal 146 1 pBG/wt ELVS/PRE 1.5-βgal 334 33 Mock 0 0

The results demonstrate clearly that the number of BHK or HT1080 cellstransfected with ELVS plasmids expressing β-galalactosidase wasincreased dramatically by inclusion of the RNA transport PRE sequence inthe vector. Further, these results indicate that one cause for thediminished heterologous gene expression levels in HT1080 cells, comparedto BHK cells, transfected with ELVS plasmid DNA is the inefficienttransport of the primary transcript from the nucleus.

In parallel with the higher frequency of reporter protein expressingHT1080 cells transfected with ELVS plasmids containing the PRE sequence,the levels of β-galalactosidase were dramatically higher in lysates fromHT1080 cells transfected with ELVS vectors containing the PRE sequence.These results are illustrated in FIG. 12B, and taken together with theresults shown in the table above, demonstrate that functional vectorreplicons are transported inefficiently from the nucleus in human cellstransfected with ELVS plasmids. Further, inclusion of the PRE sequencein the ELVS plasmid construct increases the level of heterologous geneexpression in all cells tested, demonstrating a clear relationshipbetween efficiency of cytoplasmic vector replicon transport and overallheterologous gene expression level.

Several other viral sequence elements which operate in cis to transportunspliced RNAs have also been identified. For example, a 219 bpsequence, located between nts 8022 and 8240 near the 3′ end of theMason-Pfizer monkey virus (MPMV) genome, has been shown to enable Revindependent human immunodeficiency virus type 1 (HIV-1) replication(Bray et al., PNAS 91:1256-1260, 1994). The MPMV RNA transport element,known as the constitutive transport element (CTE), is inserted into thepBG/SIN-1 ELVS 1.5-βgal plasmid by first isolating a PCR-generated 219bp fragment of MPSV from the full length genomic clone template (Sonigoet al., Cell 45:375-385, 1986), or the MPSV subgenomic clonepGEM7FZ(−)MPSV 8007-8240 (D. Rekosh, Ham-Rek Laboratories, SUNY atBuffalo, 304 Foster Hall, Buffalo, N.Y.). The amplified fragment extendsfrom base 8022-8240 of the MPSV genome. The primer sequences are givenbelow.

Forward Primer: NMPVM8021 F (SEQ. ID NO. 48)

5′ -CCTATGCGGCCGCTAGACTGGACAGCCAATGACG

Reverse Primer: EMPMV8241R (SEQ. ID NO. 49)

5′-CCTATTGGCCAGCCAAGACATCATCCGGGCAG

The primers introduce a Not I recognition site at the 5′ end of thefragment and an Eae I recognition site at the 3′ end. Not I and Eae Ihave compatible sticky ends. The Eae I recognition site is internal tothe Not I site, so Eae I cuts both sites.

The PCR fragment is digested with Eae I and cloned into Not Idigested/CLAP treated pBG/wt ELVS 1.5-βgal. The correct clone retainsthe Not I site at the 5′ terminus of the PRE and is called pBG/wtELVS/CTE 1.5-βgal.

In addition to the HBV PRE and MPSV CTE sequences, several RNA transportelements from other viral or cellular sources can be inserted into theELVS plasmid constructs, as described above. For example, some of theseelements include the HIV Rev responsive element (Malim et al., Nature,338:254-257, 1989), the HTLV 1 Rex element (Ahmed et. al, Genes Dev.,4:1014-1022, 1990), and another cis-acting sequence from simianretrovirus type 1 (Zolotukhin et al., J. Virol., 68:7944-7952, 1994). Inaddition, each of the above RNA transport elements also may beincorporated into the structural protein expression casettes, packagingcell lines, or producer cell lines described in Examples 6 and 7.

In yet another modification of prototype ELVS vectors, expression of thealphavirus replicons can be driven from an RNA polymerase I promoter.Briefly, because RNA polymerase I promoters are not tissue specific andare expressed in essentially all human cells in the body, they providean attractive alternative for plasmid DNA-directed alphavirus repliconexpression in transfected cells. For example, the human rDNA promoter(plasmid prHU3, Learned and Tjian, J. Mol. Appl. Gen., 1:575-584, 1982),has been used to construct a vector for heterologous gene expression(Palmer et al., Nuc. Acids Res. 21:3451-3457, 1993).

Thus, within one embodiment of the invention a RNA polymerase I promotercan be juxtaposed with the 5′ end of the replicon cDNA such that thefirst nucleotide transcribed in transfected cells corresponds to theauthentic alphavirus 5′ end. Identification of the RNA polymerase Ipromoter (e.g., plasmid prHU3) nucleotide at which transcriptioninitiation occurs is determined as described previously (Dubensky etal., W/O 95/07994).

All modifications described herein can be performed with ELVS constructscontaining the Sindbis virus wild-type or SIN-1 nsPs, or nsP genes fromany alphavirus. For example, all of the constructions provided inExample 5 can also be performed with plasmid pBG/SIN-1 ELVS 1.5-βgal,whose construction is described in Example 4.

Example 6 Construction of Alphavirus packaging Cell Lines

In the present invention, alphavirus packaging cell lines (PCL) areprovided, whereby the virus-derived structural proteins necessary forRNA packaging and formation of recombinant alphavirus vector particlesare encoded by one or more stably transformed structural proteinexpression cassette(s). Synthesis of these proteins preferably occurs inan inducible manner, and in particularly preferred embodiments. viatranscription of subgenomic MRNA from their native “junction region”promoter. Inducible subgenomic transcription is mediated by the inputalphavirus vector RNA itself (FIG. 13). Following primary transcriptionfrom the structural protein expression cassette(s), cytoplasmicamplification of the RNA transcript is initiated by vector-encodednonstructural proteins, and ultimately leads to transcription from thejunction region promoter and high level structural protein expression.The structural protein expression casettes may include any of thepreviously described elements of the present invention, including RNAtransport elements (e.g., HBV PRE and MPMV CTE) and splicing sequences.Such PCL and their stably transformed structural protein expressioncassettes can be derived using methods described within PCT applicationWO 95/07994, or using novel approaches described within this invention.PCL may be derived from almost any existing parental cell type,including both mammalian and non-mammalian cells. Preferred embodimentsfor the derivation of PCL are cell lines of human origin.

A. Construction of Vector-Inducible Alphavirus PCL

For example, an alphavirus structural protein expression cassette wasconstructed, whereby primary transcription from a CMV immediate earlypromoter produces an RNA molecule capable of efficient cytoplasmicamplification and structural protein expression only after translationof nonstructural replicase proteins from the vector RNA. Specifically,plasmid pDCMV-dlnsPSIN (Dubensky et al., J. Virol. 70;508-519, 1996), aDNA-based Sindbis defective helper (DH) vector, wag modified to containboth a hepatitis delta virus (HDV) antigenomic ribozyme sequence(Perotta and Been, Nature 350:434-436, 1991) for 3′-end RNA processing,and an SV40 small t antigen intron inserted within the region ofnonstructural protein gene deletion. Due to restriction siteduplications associated with insertion of the HDV ribozyme sequence, anadditional plasmid from Dubensky et al, (ibid), pDLTRSINgHDV, was usedas starting material to reconstruct the modified CMV-based DH construct.Plasmid pDLTRSINgHDV, an LTR-based Sindbis genomic clone containing theHDV ribozyme, was digested with Bgl II to remove the existing LTRpromoter and Sindbis nucleotides 1-2289 (numbering according to Strausset al., Virology 133:92-110, 1984), treated with calf intestinalalkaline phosphatase, and purified from a 0.7% agarose gel usingGENECLEAN II™ (Bio101, San Diego, Calif.). The corresponding 5′-endfragment with a CMV promoter was obtained by Bgl II digestion of theSindbis genomic clone pDCNIVSINg (Dubensky et al., ibid) andpurification from a 1% agarose gel using GENECLEAN II, and then ligatedinto the Bgl II-deleted pDLTRSINgHDV vector to generate the constructpDCMVSINgHDV. This CMV-based genomic plasmid with an HDV ribozyme wasshown to produce infectious Sindbis virus and cytopathic effect within24 hr after transfection into BHK cells. Defective helper plasmidpDCMVdlnsPSINgHDV, containing the HDV ribozyme, was then constructed byBspE I digestion and relegation under dilute conditions, to removenonstructural gene sequences between nucleotides 422 and 7054.Subsequently, the SV40 intron was synthesized by PCR and inserted intothe region of nonstructural protein gene deletion. Amplification of theSV40 intron sequence was accomplished by standard three-cycle PCR with a30 second extension time, using plasmid pBR322/SV40 (strain 776, ATCC#45019) as template and the following oligonucleotide primers that weredesigned to contain flanking BspE I or Bam HI sites.

Forward primer: BspSVSDF (5′-rest. site/SV40 intron seq.) (SEQ. ID. NO.50)

5′-TATATATCCGGA/AAGCTCTAAGGTAAATATAAAATTTTT-3′

Reverse primer: BamSVSAR (5′-rest. site/SV40 intron seq.) (SEQ. ID. NO.51)

5′-TATATAGGATCC/TAGGTTAGGTTGGAATCTAAAATACACAAAC-3′

Following amplification, the DNA fragment was purified using aQIAquick-spin PCR purification kit (Qiagen, Chatsworth, Calif.),digested with BspE I and Bam HI, purified from a 1.2% agarose gel usingMermaid™ (Bio101, San Diego, Calif.), and ligated into the defectivehelper plasmid pDCMV-dlnsPSIN, which was also digested with BspE I andBam HI, treated with calf intestinal alkaline phosphatase, and purifiedfrom a 0.7% agarose gel using GENECLEAN II, to generate the constructpDCMV-intSINrbz (FIG. 14). Plasmid pDCMV-intSINrbz, which also containsan SV40 promoter-driven neomycin resistance selectable marker on anotherportion of the plasmid, was transfected into BHK cells usingLipofectamine™ (Gibco/BRL, Gaithersburg, Md.), as described by themanufacturer. Approximately 24 hr post-transfection, the cells weretrypsinized and re-plated in media containing 600 ug/ml G418 (neomycin).The media was exchanged periodically with fresh G418-containing mediaand foci of resistant cells were allowed to grow. Cells were trypsinizedand cloned by limiting dilution in 96 well tissue culture dishes, andindividual cell clones were grown and expanded for screening.

Positive packaging activity for the individual clones was identified byLipofectin™ (Gibco/BRL, Gaithersburg, Md.)-transfection with Sindbisvector RNA that expresses a luciferase reporter gene (described inDubensky et al., ibid), harvesting the culture supernatants atapproximately 24 hr post-transfection, and assaying for the presence ofpackaged Sindbis-luciferase vector particles. In addition, initialtransfection levels were determined by harvesting the transfected celllysates using reporter lysis buffer (Promega, Madison, Wis.), andtesting for the presence of luciferase activity by using luciferinsubstrate (Promega), as described by the manufacturer. To assay forpackaged vector particles in the culture supernatants, 1 ml ofundiluted, clarified supernatant was used to infect fresh BHK cellmonolayers for approximately 18 hr. The cells were subsequently lysed asabove, and luciferase activity was determined. The presence ofluciferase activity in the infected BHK cells was confirmation ofpackaged vector particles in the transfected cell supernatants. Severalpositive cell clones harboring integrated copies of the pDCMV-intSINrbzstructural protein expression cassette, and functioning as PCL, wereidentified. Packaging data for two of the individual PCL clones (#10s-19and #10s-22), that were representative of the group, are shown in FIG.15. In addition, the titer of packaged vector particles being producedwas determined by transfecting an individual PCL clone (#10s-22) withSIN-β-gal vector RNA (described in Dubensky et al., ibid). The culturesupernatant was recovered at 48 hr post-transfection, clarified bypassage through a 0.45 mm filter, and fresh BHK monolayers were infectedwith 10-fold dilutions of the supernatant. Approximately 14 hrpost-infection, the cells were washed with PBS, fixed with 2%formaldehyde, washed again with PBS, and stained with X-gal. Vectorparticle units were then determined by counting individual blue-stainedcells. Packaged β-gal vector titers from this PCL clone wereapproximately 10⁶ infectious units/ml of supernatant. Vector-controlledinducibility of Sindbis structural protein expression was demonstratedby western blot analysis using a polyclonal rabbit antiserum specificfor the structural proteins. Positive 10s-22 PCL and negative controlBHK cell lysates were made in Lameli sample buffer, either before (U;uninduced) or after (I; induced) transfection with SIN-β-gal vector RNA.As shown in FIG. 16, the only cell lysate that showed expression ofSindbis structural proteins was the 10s-22 PCL clone after transfectionwith vector RNA. Differences in the apparent levels of expressionbetween the capsid protein and envelope glycoproteins do not reflect theactual amounts of protein being made, rather, the lower stability of theenvelope glycoproteins during the cell lysis procedure used for thisparticular experiment.

Packaging activity of the CMV-based DH construct was also highlyefficient in non-mammalian cells, for example, C6/36 mosquito cells. Theuse of such a non-mammalian parental cell type for derivation of PCL maybe particularly advantageous when the PCL are intended for subsequentuse as starting material for the generation of vector producer celllines. The advantage of this cell type is the natural ability ofalphaviruses to establish a persistent infection, without the mammaliancell-associated phenotype of inhibition of host macromolecular synthesisand resulting cytopathic effect (CPE). Thus a DNA-based alphavirusvector (Examples 4 and 5), with an appropriate selectable marker may bestably transformed into mosquito or other non-mammalian cell-derivedPCL. FIG. 17A shows that both a DNA-based luciferase reporter vector andDH helper vector expressing Sindbis structural proteins, under thecontrol of the CMV promoter, were fully functional in C6/36 cells, asdemonstrated by luciferase vector packaging.

B. Construction of PCL with operably-linked selection marker

In other embodiments of the present invention, a selectable marker isoperably linked to transcription of the alphavirus structural proteinexpression cassette. In preferred embodiments, this operable linkage isaccomplished either by insertion of the marker into the region ofnonstructural protein gene deletion, as a fusion with remaining nsP1amino acids, or by insertion downstream of the structural protein genes,under the translational control of an internal ribosomal entry site(IRES) sequence. Again, amplification of the primary structural proteingene mRNA transcript and induction of structural protein expression iscontrolled by the input vector RNA molecule and its synthesizednonstructural proteins.

Specifically, for construction of the structural protein expressioncassette, plasmid pBGS131 (Spratt et al., Gene 41:337-342, 1986; ATCC#37443) was modified to remove extraneous sequences, and to render anexisting Xho I site within the kanamycin resistance gene non-functional.Plasmid pBGS131 was digested with Xho I and a synthetic double-strandedoligonucleotide linker with Xho I-compatible ends was ligated into thesite. The synthetic 12-mer oligonucleotide, shown below, was designed asa partial palindrome that would anneal to itself generating Xho I stickyends for ligation, and maintaining the kanamycin resistance gene openreading frame by inserting four in-frame amino acids.

dLXholinker (SEQ. ID. NO. 52)

5′-TCGATCCTAGGA

Insertion of this oligonucleotide resulted in a Xho I site-deletedplasmid, designated pBGS131dlXhol The plasmid was next digested withBspH I and religated to itself under dilute conditions to remove 829 bpof extraneous sequence between the ColE1 replicon and kanamycinresistance marker, generating the plasmid pBGS131dlB. The BspH I sitenext was changed to a Pac I site by digesting pBGS131dIB with BspH I,making the termini blunt with Klenow enzyme and dNTPs, and ligating withexcess Pac I linker.

Pac I linker (SEQ. ID. NO. 53)

5′-GCTCTTAATTAAGAGC

This new construct, designated pBGS131dlB-P, was further modified bydigesting with Fsp I and Pvu II to remove an additional 472 bp,including the multiple cloning site (MCS) and purifying the remainingvector from a 1% agarose gel using GENECLEAN II. A replacement MCS wasinserted into the modified vector by annealing two complimentaryoligonucleotides, PME.MCSI and PME.MCSII, and ligating with the linearplasmid.

PME.MCSI (SEQ. ID. NO. 54)

5′-CTGTTTAAACAGATCTTATCTCGAGTATGCGGCCGCTATGAATTCGTTTAAACGA-3′

PME.MCSII (SEQ. ID. NO. 55)

5′-TCGTTTAAACGAATTCATAGCGGCCGCATACTCGAGATAAGATCTGTTTAAACAG-3′

The new, approximately 2475 bp, cloning vector was designated pBGSVG,and contained the following multiple cloning site: <Pme I-Bgl II-XhoI-Not I-EcoR I-Pme I>. Insertion of the structural protein expressioncassette containing an operably linked selectable marker proceededstepwise, as follows. A DNA fragment comprising the 3′-end of Sindbisvirus, a synthetic A₄₀ tract, the antigenomic HDV ribozyme, and a BGHtranscription termination signal, was removed from plasmid pBG/SIN-1ELVS 1.5 (Example 5) by digestion with Not I and EcoR I, andpurification from a 1% agarose gel using GENECLEAN II. Plasmid pBGSVGalso was digested with Not I and EcoR I, purified from a 1% agarose gelusing GENECLEAN II, and ligated with the purified 3′-end/A40/HDV/BGHfragment, to generate the construct pBGSV3′. Next, an approximately 9250bp Sindbis cDNA fragment, containing the structural protein genes andmuch of the nonstructural protein-encoding region, was removed fromplasmid pDLTRSINg (Dubensky et al., ibid) by digestion with Bgl II andFsp I, and purified from a 0.7% agarose gel using GENECLEAN II. TheSindbis CDNA fragment was then ligated into plasmid pBGSV3′, which wasalso digested with Bgl II and Fsp I, treated with alkaline phosphatase,and purified from a 0.7% agarose gel using GENECLEAN II. The newconstruct was designated pBGSV3′BF. Subsequently, this construct wasdigested with Bgl II, treated with alkaline phosphatase, and purifiedwith GENECLEAN II for insertion of remaining 5′-end and nonstructuralgene sequences, along with a CMV IE promoter. The remaining sequenceswere obtained by digestion of plasmidpDCMVSINg (Dubensky et al., ibid)with Bgl II, purification of the fragment from a 1% agarose gel usingGENECLEAN II, and ligation with the linear pBGSV3′BF vector, to createthe CMV-driven Sindbis genomic construct, pBGSVCMVgen. Functionality ofthis construct for initiation of the Sindbis virus replication cycle wasdetermined by Lipofectamine-mediated transfection of pBGSVCMVgen plasmidinto BHK cells, and the observance of CPE within 24 hrpost-transfection.

Plasmid pBGSVCMVgen was subsequently used to construct a DH structuralprotein expression cassette by deleting most of the nonstructuralprotein gene sequences and inserting a neomycin resistance gene as anin-frame fusion with remaining codons of the nsP1 open reading frame.Briefly, the neomycin resistance gene was amplified by standardthree-cycle PCR from the pcDNA3 vector (Invitrogen, San Diego, Calif.),using the following oligonucleotide primers that were designed tocontain flanking BspE I and BamH I sites.

Forward primer: NEO5′FUSE (5′-rest. site/neo gene) (SEQ. ID. NO. 56)

5′-ATATATCCGGA/GTCCGGCCGCTTGGGTGGAGAGGCTA

Reverse primer: NEO3′BAM (5′-rest. site/neo gene) (SEQ. ID. NO. 57)

5′-ATATAGGATCC/TCAGAAGAACTCGTCAAGAAGGCGA

Following amplification, the DNA fragment was purified withQIAquick-spin, digested with BspE I and BamH I, purified using GENECLEANII, and ligated into plasmid pBGSVCMVgen that had also been digestedwith BspE I and BamH I, treated with alkaline phosphatase, and purifiedfrom a 0.7% agarose gel using GENECLEAN II. The resulting construct wasdesignated pBGSVCMVdlneo, and is shown schematically in FIG. 14. Theconfiguration of pBGSVCMVdlneo includes, as part of the structuralprotein expression cassette and controlled by the same CMV promoter, afusion protein comprising the initiator methionine and amino-terminal121 amino acids of nsP1 and the neomycin resistance gene lacking itsmethionine initiator codon and next ten amino acids.

Plasmid pBGSVCMVdlneo was transfected into BHK cells usingLipofectamine, as described by the manufacturer. Approximately 24 hrpost-transfection, the cells were trypsinized and re-plated in mediacontaining 600 μg/ml of the drug G418 (neomycin). The media wasexchanged periodically with fresh G418-containing media and foci ofresistant cells were allowed to grow. Cells were trypsinized and clonedby limiting dilution in 96 well tissue culture dishes, and individualcell clones were grown and expanded for screening. Positive packagingactivity for the individual clones was identified by transfecting withSindbis luciferase vector RNA and assaying for the presence of packagedSindbis-luciferase vector particles as described in the previoussection. Several positive cell clones harboring integrated copies of thepBGSVCMVdlneo structural protein gene expression cassette, andfunctioning as PCL, were identified. SNBS™-luciferase vector packagingdata for individual clones (F11, F13, F15) that are representative ofthe group, as well as the previously described 10s-22 PCL line, areshown in FIG. 18.

In addition to demonstrating functional packaging activity withSindbis-lucifease vectors, additional experiments performed using thesame PCL also showed that vectors derived from other alphaviruses alsocould be packaged. For example, both Sindbis (Dubensky et al., ibid.)and Semliki Forest (pSFV3-lacZ; GIBCO BRL, Gaithersburg, Md.) vectorRNAs expressing β-galactosidase, were transfected into the F15 packagingcell line. Approximately 48 hr post-transfection, the culturesupernatants were harvested, clarified, diluted serially, and used toinfect fresh BHK cell monolayers for determination of vector particletiters. At 18 hr post-infection, the BHK cells were fixed, stained withX-gal, and the blue-staining cells were counted, as describedpreviously. The vectortiters obtained for Sindbis β-gal wereapproximately 5×10⁶ IU/ml, while the titers for SFV β-gal wereapproximately 4×10⁶ IU/ml. These data demonstrate that the two differentalphaviruses and their corresponding vectors have similar packagingsignals and that PCL derived for the Sindbis systems described hereinare fully functional when used with another alphavirus.

Packaging activity of the pBGSVCMVdlneo construct also was highlyefficient in cells of human origin, for example, 293 cells. The use ofsuch a human parental cell type for derivation of PCL may beparticularly advantageous in the generation of complement resistantrecombinant alphavirus particles. FIG. 17B shows that both RNA andDNA-based luciferase reporter vectors were efficiently packagedfollowing transfection into G418 resistant. pBGSVCMVdlneo-transformed293 PCL, as demonstrated by supernatant transfer of luciferaseexpression into BHK cells.

Another selectable drug-resistance marker also was shown to function ina similar PCL configuration, as a fusion protein with remaining nsP1amino acids at its N-terminus. Briefly, the hygromycin phosphotranferasegene (hygromycin resistance marker, hygro′) was substituted into plasmidpBGSVCMVdlneo, in place of the existing neomycin resistance marker. Thehygro′ gene was amplified by standard three-cycle PCR from plasmid p3′SS(Stratagene, La Jolla, Calif.), using the following oligonucleotideprimers that were designed to contain flanking EcoRV and BamHI sites.

Forward primer: 5′HYGROEV (5′-rest. site/hygro gene) (SEQ. ID. NO. 112)

5′-TATATGATATC/AAAAAGCCTGAACTCACCGCGACG

Reverse primer: 3′HYGROBA (5′-rest. site/hygro gene) (SEQ. ID. NO. 113)

5′-ATATAGGATCC/TCAGTTAGCCTCCCCCATCTCCCG

Following amplification, the DNA fragment was purified withQIAquick-spin, digested with EcoRV and BamHI, purified using GENECLEAN,and ligated into plasmid pBGSVCMVdlneo that had been digested withBspEI, blunt-ended with Klenow, digested further with BamHI, treatedwith alkaline phosphatase, and purified from a 0.7% gel using Geneclean.The resulting construct was designated pBGSVCMVdlhyg.

Plasmid pBGSVCMVdlhyg was transfected into BHK cells usingLipofectamine, as described by the manufacturer. Approximately 24 hrpost-transfection, the cells were trypsinized and re-plated in mediacontaining 1.2 mg/ml of the drug hygromycin (Boehringer Mannheim). Themedia was exchanged periodically with fresh hygromycin-containing mediaand foci of resistant cells were allowed to grow into a pool.Functionality of the selected packaging cells was demonstrated bytransfecting with Sindbis luciferase vector RNA and assaying for thepresence of packaged Sindbis-luciferase vector particles as described inthe previous section. Positive results from these packaging experimentsare shown in FIG. 39.

In an alternative packaging cell line structural protein expressioncassette, the selectable marker (in this case neomycin resistance) wasinserted downstream of the Sindbis structural protein genes and underthe translational control of an internal ribosome entry site (IRES).Thus, transcription of the mRNA encoding neomycin resistance occurs bothat the genomic level (from the RSV promoter) and also from thesubgenomic junction region promoter. Additional features unique to thisconstruct include the Rous sarcoma virus (RSV) LTR promoter for primarytranscription and a tRNA^(Asp) 5′-end sequence derived from Sindbisdefective-interfering RNA clone D125 (Monroe and Schlesinger, Proc.Natl. Acad. Sci. USA 80:3279-3283, 1983). This particular PCL expressioncassette configuration was designated 987DHBBNeo, and is shownschematically in FIG. 14. Specifically, plasmid 987DHBBNeo may beconstructed stepwise using the modified plasmid vector pBGS131dlB-P(described above) as starting material. A cDNA fragment containing thejunction region promoter, the structural protein gene sequences, and3′-untranslated region+poly A is obtained by digestion of thefull-length Sindbis cDNA clone pRSINg (Dubensky et al., ibid) with BamHIand Xba I, and purification from a 0.7% agarose gel using GENECLEAN II.The Sindbis cDNA DNA fragment is ligated with plasmid vectorpBGS131dIB-P that also has been digested with BamH I and Xba I, treatedwith alkaline phosphatase, and purified from a 0.7% agarose gel usingGENECLEAN II, to generate the construct pBGSINsp.

Next, the transcription termination signal from the SV40 early region isinserted between the Sac I and Eco RI sites of pBGSINsp, immediatelydownstream of the Sindbis sequence. The SV40 viral nucleotides 2643 to2563, containing the early region transcription termination sequences,are isolated by PCR amplification using the primer pair shown below andthe pBR322/SV40 plasmid (ATCC #45019), as template.

Forward primer: FSVTT2643 (5′-rest. site/SV40 nts 2643-2613) (SEQ. ID.NO. 58)

5′-TATATATGAGCTCTTACAAATAAAGCAATAGCATCACAAATTTC

Reverse Primer: RSVTT2563 (rest. site/SV40 nts 2563-2588) (SEQ. ID. NO.59)

5′-TATATGAATTCGTTTGGACAAACCACAACTAGAATG

The primers are used in a standard three-cycle PCR reaction with a 30second extension period. The amplification products are purified withQIAquick-spin, digested with Sac I and Eco RI, purified again with theMermaid kit, and the 90 bp fragment is ligated into plasmid pBGSINspthat also has been digested with Sac I and EcoRI, treated with alkalinephosphatase, and purified from a 0.7% agarose gel using GENECLEAN II.This construction is known as pBGSINspSV.

Next the RSV promoter and Sindbis 5′-end sequences, including the DItRNA^(Asp) structure, are assembled by overlapping PCR and the entirefragment is inserted into the structural protein gene vector pBGSINspSV.In PCR reaction #1, the RSV promoter fragment is amplified by standardthree cycle PCR, with a 1 minute extension, from an RSVpromoter-containing template plasmid (e.g. pRc/RSV, Invitrogen, SanDiego, Calif.), using the following oligonucleotide primers that aredesigned to also contain a flanking Bgl II site in one primer andsequences overlapping the tRNA 5′-end in the other. The Sindbis tRNA5′-end is positioned immediately adjacent to the RSV promotertranscription start site.

Forward primer: 5′RSVpro (5′-rest. site/RSV seq.) (SEQ. ID. NO. 60)

5′-TATATAGATCTIAGTCTTATGCAATACTCTTGTAGT

Reverse primer: 3′RSVtR (5′-Sin tRNA seq/RSV seq.) (SEQ. ID. NO. 61)

5′-GGGATACTCACCACTATATCTCGACGGTATCGAGGTAGGGCACT

In PCR reaction #2, the Sindbis 5′-end plus tRNA sequence is amplifiedby standard three cycle PCR with a 1 minute extension, from templateplasmid Toto1101(5′tRNA^(Asp)) (Bredenbeek et al., J. Virol.67:6439-6446, 1993), using the following oligonucleotide primers thatare designed to also contain a flanking BamH I site in one primer andsequences overlapping the 3′RSVtR primer in the other.

Forward primer: 5′tRNASin (5′-Sindbis+tRNA seq. only) (SEQ. ID. NO. 62)

5′-GATATAGTGGTGAGTATCCCCG

Reverse primer: 3′SinBam (3′-rest. site/Sindbis seq.) (SEQ. ID. NO. 63)

5′-TATATGGATCCIAGTACGGTCCGGAGATCCTTAATCTTCTCATG

Following amplification, the DNA fragments are purified withQIAquick-spin and used together as templates in a subsequent three-cyclePCR reaction with 2 minute extension, using additional 5′RSVpro and3′SinBam primers. The resulting overlapping PCR amplicon is purifiedusing GENECLEAN II, digested with Bgl II and BamH I, and ligated intoplasmid pBGSINspSV that also has been digested with Bgl II and BamH I,treated with alkaline phosphatase, and purified from a 0.7% agarose gelusing GENECLEAN II. The resulting structural protein expressing,defective helper is designated 987DHBB.

Next, the IRES sequence from encephalomycodarditis virus (EMCV), ispositioned immediately upstream of the neomycin phosphotransferase gene,as a selectable marker, by overlapping PCR, and the entire amplicon isinserted into the Nsi I site of 987DHBB. Insertion at the Nsi I sitewill position the selectable marker immediately downstream of thestructural protein ORF. In PCR reaction #1, the EMCV IRES fragment(nucleotides 260-827) is amplified by standard three cycle PCR, with a30 second extension, from template plasmid pBS-ECAT (Jang et al., J.Virol 63:1651, 1989), using the following oligonucleotide primers thatare designed to also contain a fanking Nsi I site in one primer andsequences overlapping the neo gene in the other.

Forward primer: 5′EMCVIRES (5′-rest. site/EMCV seg.) (SEQ. ID. NO. 64)5′-TATATATGCAT/CCCCCCCCCCCCCAACG

Reverse primer: 3′EMCVIRES (5′:pcDNA+neo seq/EMCV seq.) (SEQ. ID. NO.65)

5′-CATGCGAAACGATCCTCATC/CTTACAATCGTGGTTTTCAAAGG

In PCR reaction #2, the neo resistance marker is amplified by standardthree cycle PCR with a 1.5 minute extension, from template plasmidpcDNA3 (Invitrogen, San Diego, Calif.), using the followingoligonucleotide primers that are designed to also contain a flanking NsiI site in one primer and sequences overlapping the 3′EMCVIRES primer inthe other.

Forward primer: 5′Neo/pcDNA (5′-pcDNA+neo seq. only) (SEQ. ID. NO. 66)

5′-GATGAGGATCGTTTCGCATGATTGA

Reverse primer: 3′Neo/pcDNA (3′-rest. site/neo seq.) (SEQ. ID. NO. 67)

5′-TATATATGCAT/TCAGAAGAACTCGTCAAGAAGGCGA

Following amplification, the DNA fragments are purified withQIAquick-spin and used together as templates in a subsequent three-cyclePCR reaction with 2 minute extension, using additional 5′EMCVIRES and3′Neo/pcDNA primers. The resulting overlapping PCR amplicon is purifiedusing GENECLEAN II, digested with Nsi I, and ligated into plasmid987DHBB that also has been digested with Nsi I, treated with alkalinephosphatase, and purified from a 0.7% agarose gel using GENECLEAN II.The resulting structural protein expression construct, with the IRES/neoinsert in the Sindbis 3′-untranslated region, is designated 987DHBBNeo.

To generate stable packaging cell lines, BHK cells were transfected with10 ug of plasmid 987DHBBNeo, using a standard calcium phosphateprecipitation protocol. Approximately 24 hr post-transfection, the mediawas replaced with fresh media containing 1 mg/ml of the drug G418. Afterone additional day, the cells were trypsinized and re-plated at{fraction (1/10)} density in media containing 500 ug/ml G418. Afterseveral more passages, the cells were subjected to dilution cloning andindividual clones were expanded. The ability of individual clones tofunction as packaging cell lines was determined by calcium phosphatetransfection of plasmid RSV/Sinrep/LacZ, a Sindbis DNA vector expressingβ-gal, and assaying for the presence of packaged vector particles in thesupernatants after 48 hr. The packaged vector replicons were titered bythe CPE assay described in Frolov and Schlesinger (J. Virol.68:1721-1727, 1994) and one that gave high titers of packaged particles,designated 987DH-BBNeo, was used for further characterization. Packagedvector titers were determined at 48 hr, following transfection of eitherRNA- or DNA-based Sindbis vectors expressing β-gal, using severaldifferent transfection techniques. The results were as follows:

transfection titers procedure nucleic acid added (infectious units/ml)electroporation RSVSINrep/LacZ DNA (2.5 ug) 1.5 × 10⁹/ml electroporationSINrep/LacZ RNA (2.5 ug)   6 × 10⁹/ml Lipofectamine RSVSINrep/LacZ DNA(2 ug) no packaged particles Lipofectin SINrep/LacZ RNA (2 ug) 5-6 ×10⁷/ml Calcium RSVSINrep/LacZ DNA (10 ug) 1.5 × 10⁹/ml Phosphate

In addition, SINrep/LacZ particles that were packaged using 987DH-BBcell lines subsequently were used to infect fresh BHK cell monolayersand examine both RNA and protein expression patterns. FIG. 19 shows theRNA pattern after BHK cells were infected with two differentpreparations of SINrep/LacZ particles at a MOI of 150 infectious unitsper cell (lanes 1 and 2), or wild-type Sindbis virus (lane 3), as acontrol. Seven hours post-infection, dactinomycin (1 ug/ml) and[³H]uridine (20 uCi/ml) were added, followed by harvest and analysis ofRNA 4 hr later, according to Bredenbeek et al. (J. Virol. 67:6439-6446,1993). The high MOI was used in order to detect possible recombinants.Horizontal lines to the right of the gel lanes indicate the Sindbis andβ-gal RNAs of interest. The highest molecular weight band indicates thegenomic RNA of the replicon or virus (lanes 1 and 2, SINrep/LacZ; lane 3Sindbis virus). The next two RNAs indicated are the genomic RNA of the987DH-BBNeo PCL expression cassette and the inducible subgenomicstructural protein MRNA from the same 987DH-BBNeo PCL cassette. Thepresence of the latter two bands demonstrates that the helper genomicRNA derived from the packaging cell line is also co-packaged. The nextRNA bands, those present in greatest abundance, are the subgenomic RNAsderived from either SINrep/LacZ (lanes 1 and 2) or the Sindbis virusgenome (lane 3).

Protein analysis was performed following infection of BHK 21 cells withpackaged SINrep/LacZ replicons at a MOI of 20 infectious units/cell.Fifteen hours post-infection, the cells were labeled with [³⁵S]methionine for 30 minutes, lysates made, and the proteins analyzed bySDS-PAGE. As shown in FIG. 20 (lanes 2 and 3), both beta-galactosidaseand the Sindbis virus capsid protein are labeled in the vectorparticle-infected cells, but not in uninfected cells (lane 1). Thepresence of capsid shows that some of the packaged particles alsocontain structural protein gene RNA transcripts from the PCL.

C. Construction of “split structural gene” PCL configurations

In other embodiments of the present invention, PCL are provided whereinthe alphavirus structural proteins are expressed, not as a polyproteinfrom a single mRNA, with its native post-translational processing, butrather, as separate proteins from independent mRNAs that are transcribedvia multiple cassettes. This approach is depicted schematically in FIG.21A. Such a configuration greatly minimizes the possibility ofrecombination or co-packaging events that lead to formation ofreplication-competent or infectious virus. In preferred embodiments, thecapsid protein is expressed from one stably transformed cassette and theenvelope glycoproteins are expressed together from a second stablytransformed cassette, and each is expressed in a vector-inducible mannerfrom the junction region promoter (described above).

For example, the Sindbis virus capsid protein gene was amplified fromplasmid pDLTRSINg (Dubensky et al., ibid), by standard three-cycle PCRwith a 1.5 minute extension, using the following oligonucleotide primersthat were designed to contain a flanking Xho I site and capsid proteingene initiation codon or a flanking Not I site and translation stopcodon.

Forward primer: SIN5′CXho (5′-rest. site/capsid seg.) (SEQ. ID. NO. 68)

5′-ATATACTCGAG/ACCACCACCATGAATAGAGGATTC

Reverse primer: SIN3′CNot (5′-rest. site/stop codon/capsid seq.) (SEQ.ID. NO. 69)

5′-TATATGCGGCCGC/TATTA/CCACTCTTCTGTCCCTTCCGGGGT

Following amplification, the capsid DNA fragment was purified withQIAquick-spin, digested with Xho I and Not I, purified using GENECLEANII, and ligated into the DNA-based Sindbis expression vectorpDCMVSIN-luc (Dubensky et al., ibid), that also had been digested withXho I and Not I to remove its luciferase reporter gene insert, treatedwith alkaline phosphatase, and purified from a 0.7% agarose gel usingGENECLEAN II. The resulting capsid protein expression construct wasdesignated pDCMVSIN-C. Plasmid pDCMVSIN-C was subsequently digested withBspE I to remove most nonstructural protein gene sequences, andre-ligated to itself under dilute conditions to create the DH vectorconstruct, pDCMVSINdl-C.

Alternatively, the vector backbone may be first modified to contain theRSV promoter and/or 5′-end tRNA sequences described previously for987DHBB. Specifically, this was accomplished by step-wise replacementsusing plasmid pBGSV3′ (see above) as starting material. The junctionregion promoter plus Xho I and Not I cloning sites were obtained as aluciferase reporter-containing fragment from pDCMVSIN-luc (see above).Plasmid pDCMVSIN-luc was digested with Bam HI and Fsp I, and theluciferase reporter-containing fragment was purified from a 0.7% agarosegel using GENECLEAN II. The fragment was ligated into plasmid pBGSV3′that also had been digested with Bam HI and Fsp I, and treated withalkaline phosphatase to produce a plasmid designated pBGSV3′BaFLuc. TheRSV promoter/5′-end tRNA sequence was then obtained from 987DHBB bydigestion with Bgl II and Bam HI and purification from a 1% agarose gelusing GENECLEAN II. This fragment was ligated into pBGSV3′BaFLuc thatwas similarly digested with Bgl II and Bam HI, to produce the constructpBRSV987dl-Luc, which may be used as starting material for either capsidor envelope glycoprotein expression constructs.

To generate a capsid gene expression construct with the RSV promoter andtRNA 5′-end sequence, the existing luciferase reporter gene insert wasremoved by digestion with Xho I and Not I, and replaced with aPCR-amplified capsid protein gene (see above), that also was digestedwith Xho I and Not I. The resulting construct was designatedpBRSV987dl-C. Insertion of a neomycin phosphotransferase selectablemarker into the region of nonstructural protein gene deletion wasaccomplished by digestion with BspE I and Bam HI, and replacement with aPCR-amplified neo gene (see above) that also was digested with BspE Iand Bam HI, and purified from a 1% agarose gel. The resulting constructwas designated pBRSV987dlneo-C and is shown schematically in FIG. 21B.

Plasmids pDCMVSINdl-C and pBRSV987dlneo-C, which contain neomycinresistance selectable markers, were transfected into BHK-21 cells usingLipofectamine, as described by the manufacturer. Approximately 24 hrpost-transfection, the cells were trypsinized and re-plated in mediacontaining 600 ug/ml of the drug G418 (neomycin). The media wasexchanged periodically with fresh G418-containing media and foci ofresistant cells were allowed to grow. Cells were trypsinized and clonedby limiting dilution in 96 well tissue culture dishes, and individualcell clones were grown and expanded for screening. Cells which induciblyexpressed capsid protein in response to input vector were identified bytransfecting with Sindbis luciferase vector RNA or Sindbisβ-galactosidase DNA vectors, making cell lysates approximately 24 or 48-hr post-transfection, and performing western blot analysis with arabbit anti-Sindbis polyclonal antibody. Several positive cell clonesharboring integrated copies of the capsid protein gene expressioncassette and inducibly expressing the protein were identified and areshown in FIG. 21D.

In order to demonstrate both inducibility and functionality of theexpressed capsid in the context of “split structural gene” cassettes, anadditional construct that expressed the Sindbis virus envelopeglycoproteins was generated from pDCMVSIN-luc. Briefly, the Sindbisenvelope glycoprotein genes were amplified from plasmid pDLTRSINg bystandard three-cycle PCR, with a 2.5 minute extension, and using thefollowing oligonucleotide primers that are designed to contain aflanking Xho I site and translation initiation codon in good Kozakcontext, or a flanking Not I site and the translation stop codon.

Forward primer: 5′GLYCO-X (5′-rest. site/initiation codon/glycoproteinseg.) (SEQ. ID. NO. 70)

5′-ATATACTCGAG/AGCAATG/TCCGCAGCACCACTGGTCACGGCA

Reverse primer: 3′GLYCO-N (5′-rest. site/glycoprotein seq.) (SEQ. ID.NO. 71)

5′-ATATAGGCGGCCGC/TCATCTTCGTGTGCTAGTCAGCATC

Following amplification, the glycoprotein gene DNA fragment was purifiedwith QIAquick-spin, digested with Xho I and Not I, purified usingGENECLEAN II, and ligated into the DNA-based Sindbis expression vectorpDCMVSIN-luc, that also had been digested with Xho I and Not I to removeits luciferase reporter gene insert, treated with alkaline phosphatase,and purified from a 0.7% agarose gel using GENECLEAN II. The resultingglycoprotein expression construct was designated pDCMVSIN1.5PE.

FIG. 21C is a western blot demonstrating vector controlled inducibilityof two different clonal capsid lines (9-3 and 9-9) cell lines, that weretransfected with Sindbis DNA vectors expressing the envelopeglycoproteins (1.5PE lanes) or a β-galactosidase reporter, pDCMVSIN-βgal(Dubensky et al., ibid; 1.5 β-gal lanes), or “mock” transfected (Mlanes), using Lipofectamine. Cell lysates were made at 48 hrpost-transfection, separated by SDS-PAGE, and transferred to membranes,where they were probed with a combination of antibodies specific forSindbis structural proteins and β-galactosidase. The blot clearly showsthe inducibility of capsid protein in response to the nonstructuralproteins supplied by either vector, as well as the expression ofβ-galactosidase and the envelope glycoproteins. Functionality of the“split structural gene” capsid cell lines, by complementation and vectorparticle packaging, was demonstrated by co-transfecting theβ-galactosidase and envelope glycoprotein vectors into a capsid cellline using Lipofectamine, and assaying for packaged particles in theculture supernatants. Approximately 48 hr post-transfection, thesupernatants were harvested and clarified for the packaging assays andvector titer determination. In addition, the cells were lysed usingLameli sample buffer and examined by western blot analysis withpolyclonal anti-Sindbis antibody, demonstrating expression of bothcapsid protein and the vector supplied envelope glycoproteins. Thesupernatants were then tested for the presence of packaged vectorparticles by infecting naive BHK cells for approximately 18 hr, andstaining for β-gal reporter gene expression, as described previously inthis example. Functionality of the cell lines for complementation andpackaging was demonstrated by the observance of blue-stained β-galexpressing cells.

To generate stable “split structural gene” PCL that have separate vectorinducible expression cassettes for both capsid protein and the envelopeglycoproteins, any of the above described capsid cell lines may be used,in conjunction with an additional envelope glycoprotein expressionconstruct that contains a different selectable marker (for example,hygromycin B resistance). In one example, pBRSV987dl-Luc was used asstarting material to generate a glycoprotein gene expression constructwith the RSV promoter and tRNA 5′-end sequence. The existing luciferasereporter gene insert of pBRSV987dl-Luc was removed by digestion with XhoI and Not I, and replaced with a PCR-amplified glycoprotein gene(pE2/E1) product (see above), that also was digested with Xho I and NotI, and purified from a 0.7% agarose gel. The resulting construct wasdesignated pBRSV987dl-Glyco. Insertion of a hygromycinphosphotransferase selectable marker into the region of nonstructuralprotein gene deletion was accomplished by digestion of plasmidpBRSV987dl-Glyco with BspE I, blunt-ending with Klenow, and furtherdigesting with Bam HI. The hygromycin insert was obtained as aPCR-amplified product (see above) that was digested with EcoR V and BamHI, and ligated into the prepared pBRSV987dl-Glyco vector. This constructwas modified further to include an RNA export element. The PRE sequencewas inserted by first isolating a PCR-generated 564 bp fragment of HBVfrom the full-length genomic clone of the ADW viral strain, pAM6 (ATCCNo. 39630), as described in Example 5. Following amplification andpurification, the purified HBV PRE fragment was cloned into thepCR-Blunt (INVITROGEN, San Diego, Calif.) plasmid vector, to yield theconstruct pHBV-PRE. The HBV PRE element then was isolated from pHBV-PREby digestion with Not I and 2% agarose gel electrophoresis, and ligatedinto the hyromycin reistance marker containing construct derived frompBRSV987dl-Glyco, that was also digested with Not I and treated withCIAP, to yield the final construct, pBRSV987dlhyg-Glyco.

In certain embodiments, it may be desirable to also include atranslation enhancement element that may derived from capsid genesequences of homologous or heterologous alphaviruses. For inclusion of aRoss River virus translation enhancer, an appropriate sequence may beobtained from the DH-BB CΔ3rrv construct described in example 8.Specifically, DH-BB CΔ3rrv was digested with Bam HI and Bsi WI, and afragment containing the junction region promoter, Ross River virustranslation enhancer, and the amino terminal sequences of the pE2 gene,was isolated using a 1.2% agarose gel and GENECLEAN II. This fragmentwas ligated into plasmid pBRSV987dlhyg-Glyco that was similarly digestedwith Bam HI and Bsi WI, to produce the expression cassette designatedpBRSV987dlhyg-rrv-Glyco, and shown schematically in FIG. 21B.

Alternatively, plasmid pBG/SIN-1 ELVS 1.5-β-gal (Example 5) may be usedas starting material, by digestion with BamH I and Fsp I to isolate thesequences comprising the junction region promoter, the β-gal reportergene, and some 3′-end sequences. The desired fragment is purified from a1% agarose gel using GENECLEAN II, and ligated into plasmidpBGSVCMVdlneo (see above) that also has been digested with Bam HI andFsp I to eliminate all structural protein gene sequences, treated withalkaline phosphatase, and purified from a 0.7% agarose gel usingGENECLEAN II. The resulting construct is designated as pBGSVCMVdlsP-luc.Plasmid pBGSVCMVdlsP-luc is next digested with Xho I and Not I to removethe luciferase reporter gene, treated with alkaline phosphatase, andpurified from a 0.7% agarose gel using GENECLEAN II, and the Xho I- andNot I-digested envelope glycoprotein PCR amplicon from above issubsequently ligated into the digested pBGSVCMVdlsP-luc vector togenerate the envelope glycoprotein expressing DH construct,pBGSVCMVdl-G. Insertion of a hygromycin resistance marker cassette intothis plasmid, as well as flanking HSV TK promoter and polyadenylationsequences, is accomplished by PCR amplification, using a standardthree-cycle protocol with 2.5 minute extension, plasmid pDR2 (Clontech,Palo Alto, Calif.) as template, and the following oligonucleotideprimers that are designed to contain flanking Pac I sites.

Forward primer: 5′HYGRO/Pro-P (5′-rest. site/pDR2 seq.) (SEQ. ID. NO.72)

5′-ACACATTAATTAA/CGATGCCGCCGGAAGCGAGAA

Reverse primer: 3′HYGRO/pA-P (5′-rest. site/DDR2 seq.) (SEQ. ID. NO. 73)

5′-ACACATTAATTAA/GTATTGGCCCCAATGGGGTCT

Following amplification, the DNA fragment is purified withQIAquick-spin, digested with Pac I, purified using GENECLEAN II, andligated into plasmid pBGSVCMVdl-G that also has been digested with PacI, treated with alkaline phosphatase, and purified using GENECLEAN II.The resulting construct is designated as pBGSVhygro-G.

Plasmid pBRSV987dlhyg-rrv-Glyco, which contains a hygromycin selectablemarker, was transfected into a clonal capsid cell line usingLipofectamine, as described by the manufacturer. Approximately 24 hrpost-transfection, the cells were trypsinized and re-plated in mediacontaining 500 ug/ml of hygromycin (Calbiochem, La Jolla, Calif.). Themedia was exchanged periodically with fresh hygromycin-containing mediaand foci of resistant cells were allowed to grow. Cells were trypsinizedand cloned by limiting dilution in 96 well tissue culture dishes, andindividual cell clones were grown and expanded for screening. Splitstructural gene PCL derived in this manner were designated C/GLYCO PCL.Positive cells which inducibly express biologically active capsidprotein and envelope glycoproteins in response to input vector wereidentified in two ways. Initially, transfer of expression experimentswere performed to demonstrate that transfected vector molecules couldinduce structural protein expression, resulting in packaging andsecretion of vector particles that could in turn be used to infect naivecells. Sindbis virus plasmid DNA vectors expressing β-galactosidase weretransfected into panels of potential C/GLYCO PCL clones derived from twoindependently selected pools (FIG. 26B, pools C and E). At 48 hrpost-transfection, supernatants were harvested and used to infect naiveBHK-21 cells for an additional 18 hr. Infected cell lysates wereharvested and enzymatic β-galactosidase activity determined. As shown inthe figure, several clones were able to package vector, resulting in thehigh level transfer of vector to naive cells. In a second experiment,transfected PCL were lysed and subjected to western blot analysis asdescribed previously. As shown in FIG. 26C, induction of both capsid andenvelope glycoprotein occurs after introduction of vector into the PCL.

D. Construction of PCL with “hybrid” structural proteins

An additional approach which may be utilized to decrease the level ofco-packaging or recombination between DH and vector RNA molecules, toenhance translation of the glycoprotein genes, or to alter the cell ortissue specificity of the packaged recombinant alphavirus vectorparticles, makes use of structural protein genes derived from otheralphaviruses or togaviruses. More specifically, numerous combinations ofalphavirus or togavirus structural protein genes for use with Sindbisvirus or different alphavirus vectors can be envisioned. For example,the capsid protein gene of Ross River virus (RRV), may be used inconjunction with the envelope glycoprotein genes of Sindbis virus(expressed from the same or a different construct), to package a Sindbisvirus-derived vector described in examples 3, 4, or 5. In addition, adeleted form of the RRV capsid protein gene may be positionedimmediately upstream of the Sindbis glycoprotein gene sequences to serveas a translational enhancer elements. As another example, the structuralproteins of Sindbis virus may be used to package Semliki Forest virusRNA vectors.

Specifically, defective helper (DH) structural protein constructs thatcontain an intact or deleted form of the RRV capsid gene (FIG. 22), plusthe Sindbis glycoprotein genes, were constructed by PCR amplification ofSindbis virus or Ross River virus sequences from plasmid templatesToto1101 (Rice et al., J. Virol. 61:3809-3819, 1987) and RR6415 (Kuhn etal., Virology 182:430-441, 1991), respectively, and incorporation ofthose sequences into the previously described DH constructs DH-BB orDH-BB(5′SIN) (Bredenbeek et al., J. Virol. 67:6439-6446, 1993), fortranscription in vitro with SP6 polymerase. The DH constructs DH-BB orDH-BB(5′SIN) different by the presence (DH-BB) or absence (DH-BB(5′SIN))of a DI-derived tRNA^(Asp) sequence at the Sindbis 5′-end. The followingtable indicates the specific PCR primer sequences and theircorresponding Sindbis or Ross River nucleotide positions, with capitalletters indicating viral nucleotides and lower case letters indicatingadditional nucleotides of restriction sites used in cloning steps orspecific point mutations. Point mutations in primer 6 (RRV Bsp) did notchange the resulting amino acid sequence.

Primers used for PCR:

1. RRV Ava: (nt.7510-7525) (SEQ. ID. NO. 74)

5′-ccacgaattcGGTCCTAAATAGATGC

2. RRV Ncil: (nt.7606-7620) (SEQ. ID. NO. 75)

5′-ccacaagcttCCGGGCGAGGCCGCC

3. RRV Nci2:(nt.7616-7630) (SEQ. ID. NO. 76)

5′-ccacggatCCCGGCGTTCCGTCC

4. RRV Apa1: (nt.8088-8102) (SEQ. ID. NO. 77)

5′-ccacaagcttGTGCACTGGGATCTG

5. RRV Apa2:(nt.8097-81 11) (SEQ. ID. NO. 78)

5′-ccacggatccGTGCACATGAAGTCC

6. RRV Bsp: (nt.8339-836 1 (SEQ. ID. NO. 79)

5′-ccacaagCTTCcGGaGTTACCCGAGTGACC

7. RRV Afl1: (nt.7820-7836) (SEQ. ID. NO. 80)

5′-ccaccttaaGCGTCGGCTTTTTCTTC

8. RRV Afl2:(nt.7892-7907) (SEQ. ID. NO. 81)

5′-ccaccttaaGAGAAGAGAAAGAATG

9. SIN Ava: (nt.7591-7594) (SEQ. ID. NO. 82)

5′-ccacaagcttGGACCACCGTAGAG

10. SIN Bam: (nt.7325-7343) (SEQ. ID. NO. 83)

5′-CCGCGTGGCGGATCCCCTG

11. SIN Bsp: (nt.8418-8433) (SEQ. ID. NO. 84)

5′-ccacgatCCGGAAGGGACAGAAG

12. SIN Bsu: (nt.8887-8902) (SEQ. ID. NO. 85)

5′-CACGGTCCTGAGGTGC

PCR reactions were performed using the primer pairs indicated below, ina standard three cycle protocol, with 30 sec extensions and Ventpolymerase, to produce the corresponding DNA fragments, which are alsoindicated below.

PCR framents:primer pairs, plasmid template

Fragment 1: pr1+pr2, RRV6415 plasmid

Fragment 2: pr3+pr4, RRV6415 plasmid

Fragment 3: pr5+pr6, RRV6415 plasmid

Fragment 4: pr3+pr7, RRV6415 plasmid

Fragment 5: pr4+pr8, RRV6415 plasmid

Fragment 6: pr9+pr10, Toto1101 plasmid

Fragment 7: pr11+pr12, Toto1101 plasmid

Following amplification the PCR products were digested with theindicated enzymes, and ligated into the pUC18 plasmid analog, pRS2,which contains additional polylinker sites and which had also beendigested with the same enzyme combinations: fragment 1 was cut with EcoRI+Hind III; fragment 2 with BamH I+Hind III; fragment 3 with BamH I+HindIII; fragment 4 with BamH I+Afl II; fragment 5 with Hind III+Afl II;fragment 6 with BamH I+Hind III; and fragment 7 with BamH I+Bsu 361. Allinsertions were sequenced to verify that artifacts had not been acquiredduring PCR.

Subsequently, the fragments were released from the pRS2 plasmids usingthe enzymes indicated below, and ligated exactly as indicated togenerate the next set of constructs. To generate FR8, fragment 6 (cut byBam HI and Ava II) was ligated with fragment 1 (cut by Ava II and NciI), fragment 2 (cut by Nci I and Hind III) and plasmid pRS2 (cut by BamHI and Hind III). To generate FR9, fragment 6 (cut by BamH I and Ava II)was ligated with fragment 1 (cut by Ava II and Nci I), fragment 4 (cutby Nci I and Afl II) and plasmid pRS2 (cut by BamH I and Afl II). Togenerate FR10, fragment 5 (cut by Afl II and ApaL 1) was ligated withfragment 3 (cut by ApaL I and Hind III) and plasmid pRS2 (cut by Afl IIand Hind III). After transformation of E. coli, plasmids were analyzedby restriction analysis and their inserts were again isolated bydigestion and used for the next steps of cloning.

The FR8 insert (cut by BamH I and ApaL I) was ligated with fragment 3(cut by ApaL I and BspM II), fragment 7 (cut by BspM II and Bsu36 I) andplasmid DH-BB (cut by BamH I and Bsu36 I). The same fragments also wereused to replace the BamH I-Bsu36 I fragment of plasmid DH-BB(5′SIN). Theresulting plasmids were designated DH-BB Crrv and DH-BB(5′SIN) Crrv,respectively (see FIG. 23). The FR9 insert (cut by BamH I and Afl II)was ligated with the FR10 insert (cut by Afl II and BspM II), fragment 7(cut by BspM II and Bsu 36 I) and plasmid DH-BB (cut by BamH I and Bsu36I). The same fragments also were used to replace the BamH I-Bsu36 Ifragment of plasmid DH-BB(5′SIN). The resulting plasmids were designatedDH-BB CΔrrv and DH-BB(5′SIN) CΔrrv (see FIG. 23).

Multiple uses for DH constructs that contain chimeric structural proteingenes are possible, and two such approaches are illustrated in FIGS. 24and 25. In FIG. 24, the intact Ross River capsid protein gene is linkedwith the Sindbis glycoprotein gene sequences (DH-BB Crrv or DH-BB(5′SIN)Crrv), as part of a defective helper construct, and co-transfected witha Sindbis reporter RNA vector replicon to demonstrate packaging intorecombinant alphavirus particles (FIG. 26). In FIG. 25, the deleted formof the Ross River capsid protein gene is linked with the Sindbisglycoprotein gene sequences (DH-BB CΔrrv and DH-BB(5′SIN) CΔrrv), as atranslational enhancer and part of the DH construct, while the Sindbiscapsid protein gene expressed from a second DH construct. Both DHconstructs are co-transfected with a Sindbis reporter RNA vectorreplicon to demonstrate packaging into recombinant alphavirus particles(FIG. 26). Additionally, the Ross River capsid protein gene may beexpressed alone from one DH construct, while the Sindbis glycoproteinsare expressed from another, for use in packaging. Using this knowledgeand the availability of several other alphaviruses from which to derivestructural protein gene sequences, a large number of different proteincombinations may be generated in similar approaches.

Alternatively, the entire complement of structural protein genes fromone alphavirus, or other members of the Togaviridae (e.g., rubellavirus) may be used to package an RNA vector derived from another, asshown above for SFV vectors and Sindbis structural proteins. Inan-alternative embodiment, the structural protein genes from Venezuelanequine encephalitis (VEE) virus may be used to package a Sindbis-virusderived vector (wild-type or displaying the phenotype described inExample 4 or 5). Such a method provides recombinant alphavirus particlescontaining vector RNAs which exhibit the desirable properties of thepresent invention, such as delayed, reduced or no inhibition of hostmacromolecular synthesis, plus, structural proteins which redirect thetropism of the recombinant particle. Venezuelan equine encephalitisvirus (VEE) is an alphavirus which exhibits tropism for cells oflymphoid origin, unlike its Sindbis virus counterpart. Therefore,Sindbis-derived vector constructs packaged by a cell line expressing theVEE structural proteins will display the same lymphotropic properties asthe parental VEE virus from which the packaging cell structural proteingene cassette was obtained.

Specifically, the Trinidad donkey strain of VEE virus (ATCC #VR-69) ispropagated in BHK-21 cells, and virion RNA is extracted using proceduressimilar to those described in Example 1. The entire structural proteincoding region is amplified by PCR with a primer pair whose 5′-ends map,respectively, to the authentic AUG translational start site, includingthe surrounding Kozak consensus sequence, and the UGA translational stopsite. The forward primer is complementary to VEE nucleotides 7553-7579,and the reverse primer is complementary to VEE nucleotides 11206-11186(sequence from Kinney et al., Virology 170:19-30, 1989). PCRamplification of VEE cDNA corresponding to the structural protein genesis accomplished using a two-step reverse transcriptase-PCR protocol asdescribed above, the VEE genome RNA as template, and the followingoligonucleotide pair, which contain flanking Xho I and Not I sites:

Forward primer: VEE 7553F (5′-rest. site/VEE capsid seq.) (SEQ. ID. NO.86)

5′-TATATATATCTCGAGACCGCCAAGATGTTCCCGTTCCAGCCA-3′

Reverse primer: VEE 11206R (5′-rest. site/VEE E1 glyco seq.) (SEQ. ID.NO. 87)

5′-TATATATATGC GGCCGCTCAATTATGTTTCTGGTTGGT-3′

Following PCR amplification, the approximately 3800 bp fragment ispurified from a 0.7% agarose gel using GENECLEAN II, and digested withXho I and Not I. The resulting fragment is then ligated into theDNA-based Sindbis expression vector pDCMVSIN-luc (see above), that alsohas been digested with Xho I and Not I to remove its luciferase reportergene insert, treated with alkaline phosphatase, and purified from a 0.7%agarose gel using GENECLEAN II. The resulting VEE structural proteinexpression construct is designated pDCMV-VEEsp. Plasmid pDCMV-VEEspsubsequently is digested, under limiting partial digest conditions, withBspE I to remove most nonstructural protein gene sequences andre-ligated to create the structural protein-expressing DH vectorconstruct. pDCMV-VEEdl.

Plasmid pDCMV-VEEdl, which also contains a neomycin resistance marker,is transfected into BHK cells using Lipofectamine, as described by themanufacturer. Approximately 24 hr post-transfection, the cells aretrypsinized and plated in media containing 600 μg/ml of the drug G418.The media is exchanged periodically with fresh G418-containing media andfoci of resistant cells are allowed to grow. Cells are trypsinized andcloned by limiting dilution in 96 well tissue culture dishes, andindividual cell clones are grown and expanded for screening. Cells whichinducibly express VEE structural proteins in response to input vectorare identified by transfecting with Sindbis luciferase vector RNA, andassaying for VEE structural protein expression in cell lysates orpackaged luciferase vector in the supernatants, as described previously.Structural protein genes obtained from variants of VEE, or otheralphaviruses and their variants differing in tissue tropism, also areuseful when following this approach. In addition, each of the variousstructural protein gene expression cassette configurations described inthis example, including split structural gene PCL, may be used.

E. Production of packaged alphavirus vectors from PCL

Alphavirus derived PCL described throughout this example may be used ina number of different ways to produce recombinant alphavirus particlestocks, and include the introduction of vector by either transfection ofRNA or DNA molecules, infection with previously produced packagedvector-containing particles, or the intracellular production of vectorfrom stably transformed expression cassettes. The utility of alphavirusPCL for the production of vector particles is demonstrated first with areporter vector construct, and later may be applied to any other vectorconstructs which express a desired heterologous sequence. For example, astock of packaged Sindbis-β-gal vector particles is obtained byelectroporation of approximately 10⁷ alphavirus C/GLYCO or other PCLcells (see above, and for example. FIGS. 15, 17, 18) with 5-10 ugpKSSIN-1-BV-β-gal or -luciferase RNA (Example 4) or pBG/SIN-1 ELVS1.5-β-gal or -luciferase DNA (Example 5), using the procedure describedin (Liljestrom and Garoff, Bio/Technology 9:1356-1361, 1991). Thetransfected PCL are incubated at a desired temperature (e.g., 37° C.),and at approximately 48 hr. post-transfection, the supernatants areharvested and clarified by passage through a 0.45 micron filter.Additional formulation may be performed using parameters illustrated inthe detailed description of this invention.

Alternatively, a stock of packaged recombinant alphavirus particles(obtained using PCL as above, or by co-transfection of vector and DHconstructs) is used to infect a naive culture of PCL, for furtheramplification. For example, 5×10⁷ of alphavirus C/GLYCO or other PCLcells are infected with a stock of packaged pKSSIN-1-BV-β-gal vector atan approximate multiplicity of infection (MOI) of 1 infectiousunit/cell. Upon reaching the cell cytoplasm, the particle delivered RNAvector is autocatalytically amplified and packaged into additionalprogeny particles. After incubation at the desired temperature (e.g.,37° C.) for approximately 48 hr., the culture supernatants areharvested, clarified by passage through a 0.45 micron filter, andformulated as desired.

In still another method which exploits the ability of PCL to furtheramplify packaged recombinant alphavirus vector particles, stocks ofpackaged particles are used to infect naive cultures of PCL to create aworking cell bank of vector-containing PCL (vector producing cells, FIG.27), which may subsequently be used to seed another naive culture ofPCL. For example, such a working cell bank is obtained by infection ofalphavirus C/GLYCO or other PCL cells with the packaged vector stock ata M.O.I.=5. Approximately 2-3 hr post-infection, the vector containingPCL are gently detached and cell number is determined. The vectorcontaining PCL may now be used directly, or aliquoted and stored inliquid N₂ as a vector producing cell bank. When desired, the cells areseeded directly into a previously growing culture of naive alphavirusC/GLYCO or other PCL at a ratio of approximately 1 vector producing cellper 1000 fresh PCL, for production of large quantities of high titerpackaged vector particles. Aliquots of culture supernatant are harvestedat various times post-coculture to determine the time of maximalrecombinant alphavirus particle production, and that time is chosen forfurther harvest, purification and formulation, as described above. Thesame sequential amplification methodology using vector producing cellsalso is useful for large-scale production of any desired recombinantprotein (FIG. 27). For the production of recombinant protein,supernatants or cell lysates may be harvested. depending on the natureof the recombinant protein.

In yet another method for producing high titer or large scale stocks ofpackaged recombinant alphavirus particles, the desired expression vectoris introduced into 1-5% of a naive alphavirus PCL culture bytransfection of in vitro transcribed RNA or plasmid DNA vector using acommonly accepted reagent or method (for example. Lipofectin orLipofectamine, respectively, or infection with vector particles at lowMOI [≦0.1]), as described herein. The recombinant vector particlesproduced by the initial cells, into which vector was introduced,subsequently infect other naive packaging cells in the culture, which inturn, produce even more packaged particles. This process of temporalamplification continues until packaged recombinant alphavirus particlesare produced in all cells of the PCL culture.

The amplification process is demonstrated in FIGS. 26D and 28. ELVS1.5-β-gal plasmid DNA was transfected into 987DHBBNeo packaging cells orinto BHK-21 cells, and the levels of β-galactosidase present in celllysates was measured, as described previously, at the indicated timespost-transfection. In BHK-21 cells, the level of β-galactosidaseexpression reached a maximum by approximately 48 hpt, and plateaued. Incontrast, the level of β-galactosidase expression continued to increaseover a longer period of time in the ELVS 1.5 β-gal transfected987DHBBNeo PCL culture, reflecting the recombinant vector particleamplification process, and the ultimate expression of β-galactosidase inall of the cells of the culture. Further, infection of split structuralgene PCL with Sindbis vector particles (FIG. 26D) also resulted inparticle amplification. In all cases, stocks of recombinant alphavirusvector particles may be formulated so as to be pharmaceuticallyacceptable, using any of the methods described herein.

Example 7 Construction of Alphavirus Producer Cell Lines

The generation of alphavirus PCL, as described above, coupled with theconstruction of DNA-based alphavirus vectors exhibiting reduced,delayed, or no inhibition of host cell macromolecular synthesis(Examples 1,2, 4 and 5), provides a relatively straightforward mechanismto derive alphavirus vector producer cell lines. In certain embodimentsof the present invention, the vector producer cell lines contain one ormore stably transformed structural protein gene expression cassettes,and also alphavirus RNA expression vector molecules with the abovephenotype, that are transfected, transduced, or intracellularlyproduced, leading to the production of packaged vector particles. Inpreferred embodiments, an RNA vector replicon is producedintracellularly from a stably transformed DNA molecule (eukaryoticlayered vector initiation system) that exists in either an integratedform or as an episomal DNA, with transcription of vector RNAs beingcontrolled inducibly by one or more stimuli provided at a desired time.This type of alphavirus producer cell line configuration essentiallyprovides a cascade of events that include: inducible production ofvector RNA and resulting autocatalytic cytoplasmic amplification of theRNA, the induction of high level structural protein expression byvector-supplied nonstructural proteins, the packaging of vector RNA bythe expressed structural proteins, and the release of packaged vectorparticles. Tightly regulated, inducible expression of vector RNA fromthe DNA molecule, once producer cell population reaches as desirednumber, is preferred, due to the potential for low level cytotoxicity ofvector replication, or the necessity to control nonstructural proteinsynthesis, as it relates to the regulation of positive strand versusnegative strand vector RNA ratios.

A. Alphavirus DNA Vectors with Single Level Regulation

In certain embodiments of the present invention, a DNA-based alphavirusvector is provided, wherein in vivo transcription of an alphavirusvector RNA molecule that is capable of autocatalytic amplificationoccurs from a promoter which is regulatable by applying a stimulus at adesired time. Such a DNA-based alphavirus vector subsequently may bestably transformed into an alphavirus packaging cell line (PCL) tocreate an inducible alphavirus producer cell line. The producer cellline configuration described herein, is therefore, a “feed-forward”system in which: 1) a stimulus is applied to the cell, resulting inefficient transcription of alphavirus vector RNA; 2) the vector RNAreplicates autocatalytically and produces nonstructural proteins; 3) thenonstructural proteins stimulate amplification of the structural proteinexpression cassette mRNAs and high level structural protein expression;and 4) the structural proteins interact with the vector RNA and resultin the subsequent packaging of recombinant alphavirus particles whichare released into the culture media. Any previously described alphavirusPCL, which is stably transformed with one or more inducible alphavirusstructural protein expression cassettes, may serve as the parental linewith which to derive the producer cell line.

For example, a tetracycline-responsive promoter system (Gossen andBujard, Proc. Natl. Acad. Sci. 89:5547-5551, 1992) may be utilized forinducible transcription of an alphavirus vector RNA. as depicted in FIG.29. In this system, the expression of a tetracycline repressor andHSV-VP16 transactivator domain, as a “fusion” protein (rTA), stimulatesin vivo transcription of the alphavirus vector RNA by bindingspecifically to a tetracycline operator sequence (tetO) locatedimmediately adjacent to a minimal “core” promoter (for example, CMV).The binding and transactivation event is reversibly blocked by thepresence of tetracycline, and may be “turned on” by removingtetracycline from the culture media. As uninduced basal levels oftranscription will vary among different cell types, other differentminimal core promoters (for example HSV-tk) may be linked to thetetracycline operator sequences, provided the transcription start siteis known, to allow juxtaposition at or in the immediate proximity ofalphavirus vector nucleotide 1.

The rTA transactivator is provided by an additional expression cassettealso stably transformed into the same cell line; and in certainembodiments, the rTA expression cassette may itself be autoregulatory.The use of an autoregulatory rTA expression cassette circumventspotential toxicity problems associated with constitutive high levelexpression of rTA by linking expression to transcriptional control bythe same tetO-linked promoter to which rTA itself binds. This type ofsystem creates a negative feedback cycle that ensures very little rTA isproduced in the presence of tetracycline, but becomes highly active whenthe tetracycline is removed (FIG. 29). Such an autoregulatory rTAexpression cassette is provided in plasmid pTet-tTAk (Shockett et al.,Proc. Natl. Acad. Sci. USA 92:6522-6526, 1995).

Functionality of such a tetracycline-regulated DNA-based alphavirusvector is demonstrated by constructing a modified SIN-1-derivedluciferase plasmid vector, which is driven by a tetracyclineoperator/CMV minimal promoter. Using plasmids pBG/SIN-1 ELVS1.5-luc(Example 4) and pBGSV3′ (Example 6) as starting material, anapproximately 7200 bp fragment, including much of the SIN-1nonstructural-encoding region, the junction region promoter andluciferase reporter gene, and a portion of the 3′-UTR, is isolated bydigestion of pBG/SIN-1 ELVS 1.5-luc with Bgl II and Fsp I, andpurification from a 0.7% agarose gel using GENECLEAN II. The 7200 bpfragment is subsequently ligated into plasmid pBGSV3′ that has also beendigested with Bgl II and Fsp I, treated with alkaline phosphatase, andpurified from a 0.7% agarose gel using GENECLEAN II. The resultingconstruct is designated pBGSVdiB/SIN1-luc. Insertion of the remainingsequences, which include the heptamerized tetracycline operator andminimal CMV promoter (tetO/CMV) linked to Sindbis nucleotides 1-2289,such that transcription will initiate with one additional nonviralnucleotide 5′ of Sindbis nucleotide 1, is accomplished by overlappingPCR. In PCR reaction #1, the approximately 370 bp tetO/CMV portion ofthe sequence is amplified by standard three-cycle PCR with a 30 secondextension from template plasmid pUHC13-3 (Gossen an* Bujard, ibid) usingthe following oligonucleotide primers that are designed to also containflanking Bgl II and Asc I sites on one primer and sequences overlapping5′-Sindbis nucleotides on the other.

Forward primer: 5′BAtetOF (5′-rest. sites/tetO nts.) (SEQ. ID. NO. 88)

5′-TATATAGATCTGGCGCGCC/TTACCACTCCCTATCAGTGATAG-3′

Reverse primer: 3′CMVpro/SINR (5′-Sindbis nts./CMV nts.) (SEQ. ID. NO.89)

5′-TACGCCGTCAAT/ACGGTTCACTAAACGAGCTCTGC-3′

In PCR reaction #2, the 2289 bp Sindbis 5′-end portion of the sequenceis amplified by standard three-cycle PCR with a three minute extension,from template plasmid pKSRSIN-1 (Example 1), using the followingoligonucleotide primers that are designed to also contain sequencesoverlapping the CMV promoter nucleotides on one primer.

Forward primer: CMVSIN5′endF (5′-CMV nts./Sindbis nts.) (SEQ. ID. NO.90)

5′-TAGTGAACCGT/ATTGACGGCGTAGTACACACTATT

Reverse primer: SIN2400R (all Sindbis nts.) (SEQ. ID. NO. 91)

5′-CGTTGAGCATAACCGAATCTAC

Following amplification, the DNA fragments are purified withQIAquick-spin and used together as templates in a subsequent three-cyclePCR reaction with 3.5 minute extension, using additional 5′BAtetOF andSIN2400OR primers. The resulting overlapping PCR amplicon ofapproximately 2660 bp is purified using GENECLEAN II, digested with BglII, and ligated into plasmid pBGSVdIB/SIN1-luc that has also beendigested with Bgl II, treated with alkaline phosphatase, and purifiedfrom a 0.7% agarose gel using GENECLEAN II. The resulting construct isdesignated ptetSIN1-luc. Vector constructs containing other heterologoussequences-of-interest are generated using a similar approach, or bydirect cloning into the Xho I and/or Not I sites. Subsequently, aselectable E. coli gpt gene (xanthine-guaninephosphoribosyltrans-ferase) expression cassette is generated andinserted into the unique Pac I site of plasmid ptetSIN1-luc, to providean additional selectable marker. First, a fragment containing the SV40promoter linked to a gpt gene open reading frame is amplified fromplasmid pMAM (Clontech, Palo Alto, Calif.) by standard three-cycle PCRwith a 2 minute extension, and using the following oligonucleotideprimers that are designed to contain upstream flanking Sac I and Pac Isites and a downstream Sac I site.

Forward primer: SV40proSPF (5′-rest. sites/SV40 promoter seq.) (SEQ. ID.NO. 92)

5′-ATATAGAGCTCTTAATTAA/TCTTIGTGAAGGAACCTTACTTC

Reverse primer: 3′ECgptR (5′-rest. site/ot gene seq.) (SEQ. ID. NO. 93)

5′-ATATAGAGCTC/AGGCGTTGAAAAGATTAGCGACCG

Following amplification, the SV40 promoter/gpt gene DNA fragment ispurified with QIAquick-spin, digested with Sac I, purified usingGENECLEAN II, and ligated into plasmid pBGS131 dlXhol-BGHTT (Example 5)that also had been digested with Sac I, treated with alkalinephosphatase, and purified from a 0.7% agarose gel using GENECLEAN II.Clones with proper orientation of the insert are identified byrestriction analysis. This configuration positions the promoter and gptgene immediately adjacent to a bovine growth hormone transcriptiontermination signal. The resulting gpt expression construct is designatedpBGS131 dlXhoI-gpt. Next the entire expression cassette is amplifiedfrom plasmid pBGS131 dlXhoI-gpt by standard three-cycle PCR with a 2minute extension, and using the following oligonucleotide primers thatare designed to contain flanking Pac I sites.

Forward primer: SV40proSPF as shown above (SEQ. ID. NO. 92)

Reverse primer: BGHTTpacR (5′-rest. site/BGH seq.) (SEQ. ID. NO. 94)

5′-TATATATTAATTAA/ATAGAATGACACCTACTCAGACAATGCGATGC

Following amplification, the gpt-gene expression cassette fragment ispurified with QIAquick-spin, digested with Pac I, purified usingGENECLEAN II, and ligated into the tet-inducible alphavirus vectorconstruct ptetSIN1-luc that also had been digested with Pac I, treatedwith alkaline phosphatase, and purified from a 0.7% agarose gel usingGENECLEAN II. The resulting construct is designated ptetSIN1gpt-luc.

For construction of an initial tetracycline-inducible alphavirus vectorproducer cell line, the ptetSIN1gpt-luc construct and a tetracyclinerepressor/VP16 transactivator (rTA) expression cassette are stablytransformed into the desired alphavirus PCL. For example, alphavirusC/GLYCO PCL cells (from above) are stably transformed with plasmidpTet-tTAk (see above) by cotransfection with another plasmid encoding aselectable marker. Plasmids pTet-tTAFk and pSV2-His, encoding ahistidinol dehydrogenase marker (Schatz et al., 1989, Cell59:1035-1048), are co-transfected into C/GLYCO PCL cells (or other PCL)at a molar ratio of 40:1 respectively, using Lipofectamine, as describedby the manufacturer. Approximately 24 hours post-transfection, the cellsare trypsinized and re-plated in media containing histidinol and 0.5ug/ml tetracycline. The media is exchanged periodically with freshdrug-containing media, and foci of resistant cells are allowed to grow.Cells are trypsinized and cloned by limiting dilution in 96 well tissueculture dishes, and individual cell clones are grown are expanded forscreening. Positive pTet-tTAk-containing packaging cell clones,designated C/GLYCO/TAk cells, are identified by transfecting theluciferase reporter plasmid pUHC13-3 (Gossen and Bujard, ibid), underthe control of a tetO/promoter, in both the presence or absence oftetracycline. In the absence of tetracycline, positive C/GLYCO/TAk PCLcells will provide induction from the tetO/promoter and inducible, highlevels of luciferase.

Subsequently, the DNA-based alphavirus vector construct ptetSIN1gpt-lucis stably transfected into the C/GLYCO/TAk cells using Lipofectamine, asdescribed by the manufacturer. Approximately 24 hr post-transfection,the cells are trypsinized and re-plated in selection media, optimizedfor the particular cell type (DMEM+10% dialyzed fetal calf serum; 250ug/ml xanthine; 15 ug/ml hypoxanthine; 10 ug/ml thymidine; 2 ug/mlaminopterin; 25 ug/ml mycophenolic acid), and containing 0.5 ug/mltetracycline. The media is exchanged periodically with fresh selectionmedia, and foci of resistant cells are allowed to grow. Cells aretrypsinized and cloned by limiting dilution in 96 well tissue culturedishes, and individual cell clones are grown are expanded for screening.Positive producer cell lines, stably transformed with ptetSIN1gpt-luc,are identified by removing tetracycline from the media for at least 24hr and testing for luciferase in cell lysates and also testing forpackaged luciferase vector in the culture supernatants, as describedpreviously.

B. Alphavirus DNA Vectors With Two Level Regulation

In preferred embodiments, it may be desirable to construct a DNA-basedalphavirus vector (wild-type or with the desired phenotype of reduced,delayed or no inhibition of host macromolecular synthesis), whether intranscription of the RNA vector molecule, capable of autocatalyticamplification, occurs from a promoter which is very tightly controlledby two levels of regulation to eliminate all basal levels oftranscription. Such an approach may combine one inducible component(e.g. the tet system from above) with a reversible transcriptionalsilencing component. For example, the KRAB repression domain of acertain zinc finger protein may be used.

Briefly, KRAB (Krüppel-associated box) domains are highly conservedsequences present in the amino-terminal regions of more than one-thirdof all Krüppel-class Cys₂His₂ zinc finger proteins. The domains containtwo predicted amphipathic α-helicies and have been shown to function asDNA binding-dependent RNA polymerase II transcriptional repressors (forexample, Licht et al., Nature 346: 76-79, 1990). Like othertranscription factors, the active repression domain and the DNA-bindingdomain are distinct and separable. Therefore, the repression domain canbe linked as a fusion protein to any sequence specific DNA bindingprotein for targeting. Ideally, the DNA binding protein component can bereversibly prevented from binding in a regulatable fashion, thus turning“off” the transcriptional silencing. For example. within one embodimentthe KRAB domain from human Koxl (Thiesen; New Biol. 2:363-374, 1990) isfused to the DNA-binding lactose (lac) repressor protein, forming ahybrid transcriptional silencer with reversible, sequence-specificbinding to a lac operator sequence engineered immediately adjacent tothe tet-responsive promoter (FIG. 30). In this configuration,constitutive expression of the lac repressor/KRAB domain fusion (rKR)will result in binding to the lac operator sequence and the eliminationof any “leaky” basal transcription from the uninduced tet-responsivepromoter. When vector expression is desired and tetracycline is removedfrom the system, IPTG is added to prevent rKR-mediated transcriptionalsilencing.

In addition, the KRAB domains from other zinc finger proteins, forexample, ZNF133 (Tommerup et al., Hum. Mol. Genet. 2:1571-1575, 1993),ZNF91 (Bellefroid et al., EMBO J. 12:1363-1374, 1993), ZNF2 (Rosati etal., Nucleic Acids Res. 19:5661-5667, 1991), and others, as well asother transferable repressor domains, for example, Drosophila en or evegenes (Jaynes and O'Farrell, EMBO J. 10:1427-1433, 1991; Han and Manley,Genes Dev. 7:491-503, 1993), human zinc finger protein YY1 (Shi et al.,Cell 67:377-388, 1991). Wilms' tumor suppressor protein WT1 (Madden etal., Science 253:1550-1553, 1991), thyroid hormone receptor (Baniahiladet al., EMBO J. 11:1015-1023, 1992), retinoic acid receptor (Baniahimadet al., ibid), Kid-1 (Witzgall et al., Proc. Natl. Acad. Sci, USA91:4514-4518, 1994), are readily used in such a system. Furthermore, thelac repressor/lac operator component of this system may be substitutedby any number of other regulatable systems derived from other sources,for example, the tryptophan and maltose operons, GAL4, etc.

Specifically, an expression cassette that contains the lac repressor(lacl) protein fused to the KRAB domain of human Koxl, with a linkednuclear localization sequence (NLS; Pro-Lys-Lys-Lys-Arg-Lys (SEQ. ID.NO. 100); Kalderon et al., Cell 39:499-509, 1984) to more efficientlydirect the protein back to the nucleus, is constructed by overlappingPCR. In PCR reaction #1, the approximately 1100 bp lacI sequence isamplified by standard three-cycle PCR with a 1.5 minute extension, fromtemplate plasmid p3′SS (Stratagene, La Jolla, Calif.), using thefollowing oligonucleotide primers that are designed to also contain aflanking Xho I site and AUG start codon in good translation initiationcontext on the upstream primer, and the SV40 large-T-antigen nuclearlocalization sequence on the other.

Forward primer: LacI5′F (5′-rest.-site/AUG+lacI sequence) (SEQ. ID. NO.95)

5′-ATATACTCGAGTAGCA/ATGGTGAAACCAGTAACGTTATAC

Reverse primer: LacI3NLSR (5′-NLS/lacI sequence) (SEQ. ID. NO. 96)

5′-GCCCTTTCTCTTCTTTTTTGG/CTGCCCGCTTTCCAGTCGGGAAAC

In PCR reaction #2, the an approximately 400 bp amplicon, comprising theamino-terminal 121 residue KRAB domain of human Kox1 is amplified bystandard three-cycle PCR with a one minute extension, from templateplasmid pKox1 (Thiesen, New Biol. 2:363-374, 1990), using the followingoligonucleotide primers that are designed to also contain sequencesoverlapping NLS and lacI on one primer and a Sac I restriction site andstop codon on the other.

Forward primer: KRAB5′F (5′-NLS+lacI overlap sequence/KRAB sequence)(SEQ. ID. NO. 97)

5′-CCAAAAAAGAAGAGAAAG/GGCGGTGGTGCTTTGTCTCCT

Reverse primer: KRAB3′R (5′-rest. site+stop codon/KRAB sequence) (SEQ.ID. NO. 98)

5′-ATATAGAGCTCTTA/AACTGATGATTTGATTTCAAATGC

Following amplification, the DNA fragments are purified withQIAquick-spin and used together as templates in a subsequent three-cyclePCR reaction with 2.5 minute extension, using additional LacI5′F andKRAB3′R primers. The resulting overlapping PCR amplicon of approximately1500 bp is purified using GENECLEAN II, digested with Xho I and Sac I,and ligated into the eukaryotic expression vector plasmid pEUK-C1(Clontech, Palo Alto, Calif.) that has also been digested with Xho I andSac I, treated with alkaline phosphatase, and purified from a 0.7%agarose gel using GENECLEAN II. The resulting lacI/KRAB expressionconstruct is designated pEUK-rKR.

To generate stable PCL transformants containing the lacI/KRAB expressioncassette, an alphavirus PCL which has already been selected fortransformation with an rTA tet/transactivator fusion protein cassette isused for starting material. For example, alphavirus C/GLYCO/TAk PCLcells (from above) are stably transformed with plasmid pEUK-rKR bycotransfection with another plasmid encoding a selectable marker.Plasmids pEUK-rKR and pPUR, encoding a puromycin acetyltransferaseselectable marker (Clontech), are co-transfected into C/GLYCO/TAk PCLcells (or other PCL) at a molar ratio of 40:1, respectively, usingLipofectamine, as described by the manufacturer. Approximately 24 hrpost-transfection, the cells are trypsinized and re-plated in mediacontaining 5 ug/ml puromycin and 0.5 ug/ml tetracycline. The media isexchanged periodically with fresh drug-containing media, and foci ofresistant cells are allowed to grow. Cells are trypsinized and cloned bylimiting dilution in 96 well tissue culture dishes, and individual cellclones are grown are expanded for screening. PositivepEUK-rKR-containing packaging cell clones, designated C/G/TAk/rKR cells,are identified by immunostaining with a polyclonal antiserum specificfor lacl (Stratagene, La Jolla, Calif.).

Next, specific lac operator (lacO) sequences must be inserted into thedesired ptet-based alphavirus vector (see above). For example, vectorconstruct ptetSIN1gpt-luc is modified to contain multiple copies of lacOby using a synthetic oligonucleotide linker. The LacO oligonucleotide isdesigned to contain a symmetric lacO sequence, including the full 22 bppalindromic operator sequence (Simons et al., Proc. Natl. Acad. Sci. USA81:1 624-1628, 1984; Sadler et al., Proc. Natl. Acad. Sci. USA80:6785-6789, 1983), and flanking Asc I sites when self-annealed into adouble-stranded molecule.

LacOsymA (SEQ. ID. NO. 99)

5′-CGCGCCGAATTGTGAGCGCTCACAATTCGG

The LacOsymA oligo is self-annealed to form a Asc I “sticky-ended” DNAfragment, and then ligated into plasmid ptetSIN1gpt-luc that has beendigested with Asc I, treated with alkaline phosphatase, and purifiedfrom a 0.7% gel using GENECLEAN II. Clones containing one, two, three,or more tandem copies of the LacO sequence are identified by sequenceanalysis, and given the designation pOItetSIN1gpt-luc,pOIItetSIN1gpt-luc, pOIIItetSIN1gpt-luc, etc. Individual clones withdifferent lacO copy numbers are then transfected as detailed below, andtested for the tightest level of transcriptional regulation.

To generate an alphavirus vector producer cell line, the DNA-basedpOtetSIN1gpt-luc vector constructs are stably transfected intoC/G/TAk/rKR cells using Lipofectamine, as described by the manufacturer.Approximately 24 hr post-transfection, the cells are trypsinized andre-plated in selection media optimized for the particular cell type(DMEM+10% dialyzed fetal calf serum; 250 ug/ml xanthine; 15 ug/mlhypoxanthine; 10 ug/ml thymidine; 2 ug/ml aminopterin; 25 ug/mlmycophenolic acid), and containing 0.5 ug/ml tetracycline. The media isexchanged periodically with fresh selection media, and foci of resistantcells are allowed to grow. Cells are trypsinized and cloned by limitingdilution in 96 well tissue culture dishes, and individual cell clonesare grown are expanded for screening. Positive producer cell lines,stably transformed with the pOtetSIN1gpt-luc constructs, are identifiedby the expression of luciferase (described previously) at least 24 hrafter the removal of tetracycline from the media and the addition of20mM IPTG for induction. Luciferase activity is determined both onproducer cell lysates and also after transfer-of-expression experimentsusing culture supernatants.

Additional levels of control may be incorporated by adding a third, oreven fourth, level of regulation to the promoter responsible fortranscription of the alphavirus vector molecule. Such extra level orregulation may be incorporated into the minimal promoter, and mayinvolve other inducible systems and/or cell differentiation control. Ineach of the above cases, stable transformation may be accomplished as anintegration into the host cell chromosome, or as an extrachromosomalepisome, using for example, the EBV episomal-based vector promoter (fornon-integrated).

Example 8 Methods for the Generation of AlphaVirus-derived Empty orChemical Viral Particles

As illustrated in Example 6, individual defective helper (DH) expressioncassettes can be constructed to contain elements from multiplealphaviruses or their variants. Thus, as described in Example 6, splitstructural gene DH cassettes for the expression of the viralglycoproteins can be constructed to contain the capsid and glycoproteingenes from different alphavirus species. For example, such aheterologous alphavirus glycoprotein DH cassette might contain thecapsid gene from Ross River virus (RRV), and the glycoprotein genes fromSindbis virus. In this configuration, the RRV capsid gene serves toenhance the level of translation of the glycoprotein genes.

The configurations described herein for the heterologous alphavirusglycoprotein DH cassettes are designed to improve the packaging ofvector replicons into alphavirus particles, yet diminish the possibilityof recombination, resulting in the formation of replication competentalphavirus. The heterologous alphavirus glycoprotein DH expressioncassette is a replacement of the Sindbis virus capsid gene in the DHexpression cassettes described in Example 6 (“genomic” structuralprotein gene PCL), with a heterologous alphavirus capsid gene (e.g.RRV). The second DH expression cassette in the split structural gene PCLcontains, for example, the Sindbis virus capsid gene. Thus, a splitstructural gene PCL for the generation of recombinant alphavirus vectorparticles having Sindbis virus structural proteins can be derived, forexample, with the Sindbis virus glycoprotein genes and capsid genes onindividual DH expression cassettes.

It has been shown previously that chimeric viruses containing all of thegenes of RRV, but with the capsid gene from Sindbis virus, or thereciprocal chimeric virus, do not assemble into infectious virusparticles (Lopez et. al., J. Virol. 68:1316-1323, 1994). The authorsconcluded in this report that the interaction between the carboxyterminus of glycoprotein E2 and capsid protein in virus assembly cannotoccur between the structural proteins of heterologous alphaviruses.Thus, recombinant genomes arising in the split structural genealphavirus PCLs described in Example 6, consisting of the Sindbis virusnon-structural protein genes (originating from the vector replicon), theRRV capsid gene, and the Sindbis virus glycoprotein genes, should not bereplication competent, resulting in the propagation of virus(replication competent Sindbis virus, RCSV). The packaging restrictionbetween heterologous alphavirus species permits the construction of DHcassettes comprised of the capsid gene. including the translationalenhancement element, from one alphavirus, and the glycoprotein genesfrom a different alphavirus. However, as illustrated in FIG. 31, theobservation of Lopez et. al. (ibid, that assembly cannot occur betweenthe structural proteins of heterologous alphaviruses, is incorrect.Indeed, a DH cassette consisting of the RRV capsid gene and the Sindbisvirus glycoprotein gene produces infectious virus particles. Briefly,BHK cells were co-electroporated with SINrep/Lac Z replicon (Bredenbeeket. al., J. Virol., 67:6439-6446, 1993), and DH-BB (5′ tRNA/SIN) Crrv(Example 6 and FIG. 24; RRV capsid/Sindbis virus glycoproteins) in vitrotranscribed RNAs. The electroporation and in vitro transcriptions wereperformed as described in Example 1. Following electroporation, the BHKcells were treated with dactinomycin and labeled with [³H]uridine,exactly as described in Example 1. At 18 hours post electroporation, theculture medium was collected, and clarified by centrifugation at 6, 000rpm for 10 min. The vector particles remaining in the supernatant werepelleted by ultracentrifugation, after first layering over a sucrosecushion. RNA was isolated from the BHK cells at 18 hours postelectroporation, and from the virus pellet, electrophoresed ondenaturing glyoxal agarose gels, and visualized by autoradiography,exactly as described in Example 1. The viral RNAs present in BHK cellselectroporated with SINrep/LacZ and DH-BB Crrv RNAs, and in virusparticles, are shown in FIG. 31 (lane 1, panel A, and lane 1, panel B).RNAs corresponding to the genomic and subgenomic replicative species forSINrep/LacZ and DH-BB Crrv RNAs were present in both electroporated BHKcells, and the produced virus particles. The results demonstrate, incontrast to Lopez et. al. (ibid), the formation of chimeric alphavirusparticles consisting of RRV capsid protein and Sindbis virusglycoproteins. Further, the indiscriminate packaging of genomic andsubgenomic SINrep/LacZ and DH-BB Crrv RNAs in chimeric alphavirusparticles indicates the inability of the Ross River capsid protein torecognize specifically the Sindbis virus packaging sequence, which ispresent in the nsP1 gene of the SINrep/LacZ vector replicon.

The viral proteins present in BHK cells electoporated with SINrep/LacZand DH-BB Crrv RNAs, at 18 hours post electroporation, and the producedvirus particles are given in FIG. 32 (lane 1, panel A, and lane 1, panelB). The viral-specific structural proteins in electroporated cells andthe produced chimeric alphavirus particles were indistinguishable. Thatis, FIG. 32 demonstrates clearly that virus particles produced from BHKcells electroporated with SINrep/LacZ and DH-BB Crrv RNAs contained theRRV capsid and the Sindbis virus glycoproteins E1 and E2. This resultprovides indisputable evidence that in contrast to Lopez et. al.,(ibid), there is no restriction in assembly between heterologousalphavirus capsid and glycoproteins that prevents the formation ofchimeric viral particles.

Thus, in distinct contrast to the results and discussion of Lopez et.al., the amino terminus of the RRV capsid protein is able to bind withthe heterologous Sindbis virus genome, and form infectious chimericalphavirus particles. Importantly, the previous conclusion that there isa restriction of virus assembly between heterologous alphavirus capsidproteins and glycoproteins is incorrect. The generation of chimericalphavirus particles as described here would, then, also result in theformation of RCSV in the split structural gene PCLs described above,since a recombinant genome consisting of the Sindbis virusnon-structural protein genes (originating from the vector replicon), theRRV capsid gene, and the Sindbis virus glycoprotein genes, wouldgenerate infectious virus. Alternatively, this lack of restriction ofpackaging between distinct alphavirus structural proteins and vectorreplicons permits the tropism of vector particles to be modified. Forexample, Sindbis virus replicons can be packaged with the VenezuelanEquine Ecephalitis virus structural proteins, in order to generate alymphotropic recombinant vector particle.

The results described in two separate previous investigations have shownthat ablation, in vitro, of the interaction between the capsid proteinand the positive RNA-stranded genome of two icosahedral viruses havingtriangulation numbers (T)=3, turnip crinkle virus (TCV), and southernbean mosaic virus (SBMV), resulted in the disassociation of the virusparticles, and the formation of nucleic acid-free T=1 particles (Sorgeret. al. J. Mol. Biol., 191:639-656, 1986, and Erickson and Rossmann,Virology 116:128-136, 1982). In the absence of nucleic acid, T=3particles similar to wild-type virus were not formed in vitro. Owen andKuhn (J. Virol., 70:2757-2763, 1996), investigated the packagingproperties of Sindbis virus genomes containing deletions in the capsid,in order to identify the region of the capsid protein that is requiredfor dictating specificity of the encapsidation reaction, in vivo. Onemutant virus [CD(97-106)] which contained a deletion corresponding toresidues 97-106 of the capsid, encapsidated both genomic and subgenomicRNAs, indicating the domain of the capsid protein required for specificrecognition of the genomic RNA packaging signal. In yet another report,the packaging properties of Aura alphavirus were investigated (Rumenapfet. al. J. Virol., 69:1741-1746, 1995). In this study, a mechanism foralphavirus packaging that involves a capsid protein-encapsidationsequence interaction initiation complex was proposed. This mechanismproposed is based on observations by the authors, and others (includingOwen and Kuhn, ibid), in which 26S and 49S alphavirus RNAs are packagedinto T=1, T=3, T=4, and T=7 virus particles, and that empty capsidsarising during infection with alphaviruses have not been reported.

Based on the literature presented above and the discussions containedtherein, a RRV capsid gene deleted of the region corresponding to thecapsid protein domain that is required for dictating specificity of theencapsidation reaction, and, in addition, surrounding basic residuesthat bind electrostatically with viral RNA, should not be able to formstable capsid particles containing viral RNAs. Thus, the alphavirusstructural proteins expressed from a heterologous alphavirus DHcassette, consisting of this deleted RRV capsid gene and the Sindbisvirus glycoprotein genes, should not assemble into stable chimericalphavirus particles. Thus, in the split structural gene PCL discussedabove and in Example 6, a recombinant genome consisting of the Sindbisvirus non-structural protein genes, the RRV capsid gene (deleted of theregion corresponding to packaging specificity and the surrounding basicresidues), and the Sindbis virus glycoprotein genes, could not generateinfectious virus. As described in Example 6, the Sindbis virus capsidprotein is expressed from a separate DH expression cassette; thus, thethree Sindbis virus structural proteins are expressed in toto, resultingin the production of recombinant vector particles.

Nucleotides of the RRV capsid gene corresponding to predicted regions ofthe expressed protein that bind to the Sindbis virus packaging sequence(Weiss et. al., Nuc. Acids, Res., 22:780-786, 1994, and Lopez et. al.,ibid), including the basic residues which bind electrostatically withthe viral RNA, were deleted in order to construct a heterologousalphavirus capsid-glycoprotein DH that provided translationalenhancement and correct pE2-6K-E1 polyprotein processing bypost-translational cleavage, yet could not assemble stable chimericRRV/Sindbis virus particles. FIG. 33 illustrates the hydrophobicityprofiles (Kyte-Dolittle) of the RRV capsid protein, and the capsidprotein expressed from 3 individual RRV capsid gene mutants (CΔ1rrv,CΔ2rrv. and CΔ3rrv), in which varying amounts of the capsid geneencoding a lysine-rich protein that interacts with the viral packagingsequence RNA, was deleted. The lysine-rich basic region of the RRVcapsid protein is shown in FIG. 33. Further, the hydrophobicity profilesdemonstrate that this lysine-rich basic region is progressivelyeliminated in the 3 individual RRV capsid gene mutants CΔ1rrv, CΔ2rrv,and CΔ3rrv. FIG. 34 demonstrates the lysine residues eliminated in theexpressed RRV capsid protein, as a result of the deletions in mutantsCΔ1rrv, CΔ2rrv, and CA3rrv. The table shown below gives the nucleotidesdeleted in the RRV genome of constructs CΔ1rrv, CΔ2rrv, and CΔ3rrv.

Construct Deleted RRV genome nts. CΔ1rrv 7841-7891 CΔ2rrv 7796-7891CΔ3rrv 7760-7891

The RRV capsid gene deletions were constructed on the DH-BB Crrv plasmidDNA illustrated in FIG. 23, and described in Example 6. The indicatedRRV capsid gene sequences were deleted by PCR, using the primers andother cloning steps given in Example 6.

FIGS. 31 and 32, discussed above, illustrate the virus-specific RNAs(FIG. 31) and proteins (FIG. 32) synthesized in BHK cells electroporatedwith SINrep/LacZ and the DH-BB CΔ1rrv, CΔ2rrv, or CΔ3rrv RNAs, andpresent in viral particles contained in the culture fluids of thesecells. The genomic and subgenomic species were detected for both theSINrep/LacZ replicon and all the three DH-BB CΔ1rrv, CΔ2rrv, or CΔ3rrvDH RNAs in electroporated cells (FIG. 31, panel A). However, theSINrep/lacZ replicon was not packaged in vector particles in cellselectroporated with DH RNA containing deletions in the RRV capsid gene,as demonstrated by the absence of replicon genomic RNA in virusparticles (FIG. 31, panel B). Further, helper genomic and subgenomicRNAs were packaged very inefficiently and were barely visible inautoradiograms of denaturing gels (FIG. 31, panel B, lanes 3 and 4),when cells were electroporated with DH molecules containing largerdeletions of the RRV capsid (CΔ2rrv or CΔ3rrv). In contrast, whileSINrep/lacZ genomic RNA (and DH RNA in electroporations with CΔ2rrv orCΔ3rrv) was not detected in viral particles from BHK cellselectroporated with the DHs containing deletions in the RRV capsid gene,equivalent RRV capsid protein and Sindbis virus glycoprotein levels wereobserved in virus particles from cells electroporated with all DH RNAs,regardless of whether the RRV capsid gene contained deletions (FIG. 32,panels A and B). This result demonstrates that stable chimeric virusparticles not containing vector replicon, or other viral-specific RNAs,were formed in BHK cells electroporated with SINrep/lacZ genomic RNA andDH RNA, from which capsid protein unable to bind to the genomic RNA wasexpressed. The formation of stable empty heterologous alphavirusparticles is unexpected and not predicted, based on the results anddiscussions of previous investigations (Lopez et. al., J. Virol.68:1316-1323, 1994, Sorger et. al. J. Mol. Biol., 191:639-656, 1986,Erickson and Rossmann, Virology 116:128-136, 1982, and Rumenapf et. al.J. Virol., 69:1741-1746, 1995).

To determine the composition of the virus particles produced, BHK cellswere electroporated with SINrep lacZ and the DH RNAs containing variousRRV capsid gene configurations, as described herein, and were treatedsubsequently with dactinomycin, and labeled with - [³⁵S]methionine and[³H]uridine, as described in Example 1. The configuration of the viralparticles produced were determined by ultracentrifugation of clarifiedcell culture media for 2 hrs at 35, 000 rpm in a SW41 rotor over a20%-40% (w/w) sucrose gradient. The results of this study are shown inFIGS. 35-37, and demonstrate again the formation of stable emptyheterologous alphavirus particles. FIG. 35 demonstrates the relativelevels of [³⁵S]methionine and [³H]uridine incorporated into particlessynthesized in BHK cells infected at high MOI (5) with wild-type virus,Toto1101. FIG. 36 demonstrates that the relative levels of[³⁵S]methionine and [³H]uridine incorporated into particles synthesizedin BHK cells electroporated with SINrep/LacZ and DH-BB (5′ tRNA) Crrv(FIG. 23) RNAs was the same as in cells infected with wild-type virus.In contrast. FIG. 37 demonstrates that the particles produced in BHKcells electroporated with SINrep/LacZ and DH-BB (5′ tRNA) CΔ3rrvcontained very low levels of incorporated [³H]uridine. FIG. 38 is acompilation of FIGS. 35-37 , and illustrates clearly that while therelative levels of [³⁵S]methionine and [³H]uridine incorporated intoparticles were similar in. BHK cells infected or electroporated withRNAs containing wild-type alphavirus capsid genes. BHK cellselectroporated with DH RNA containing deletions of nts. 7760-7891 of theRRV capsid gene formed stable chimeric empty alphavirus particles,devoid of SINrep/LacZ RNA. Titers of empty alphavirus particles,produced in cell lines electroporated with with SINrep/LacZ RNA andDH-BB CΔ3rrv in vitro transcribed RNAs, and labeled with[³⁵S]methionine, were determined by comparison with BHK cells, infectedwith Toto 1101 wild-type virus, and labeled with [35S]methionine. Thelevel of radioactivity present in virus-containing sucrose gradientfractions from Toto 1101-infected cells was quanitated, and related tothe virus titer present in these same fractions, as determined by plaqueassay, according to the methods described in Example 1. For emptyalphavirus particle titer determinations, the level of radioactivitypresent in virus particle-containing sucrose gradient fractions from BHKcells electroporated with SINrep/LacZ RNA and DH-BB CD3rrv in vitrotranscribed RNAs, was quantitated, and related to the[³⁵S]methionine/virus titer, from Toto 1101 infected cells. The titer ofempty chimeric virus particles, containing the deleted Ross River viruscapsid and the Sindbis virus glycoproteins, produced in SINrep/LacZ RNAand DH-BB CD3rrv in vitro transcribed RNA electroporated cells was 1×10⁹particles/ml.

While the present invention has been described above both generally andin terms of preferred embodiments, it is understood that variations andmodifications will occur to those skilled in the art in light of thedescription, supra, Therefore, it is intended that the appended claimscover all such variations coming within the scope of the invention asclaimed.

Additionally, the publications and other materials cited to illuminatethe background of the invention, and in particular, to provideadditional details concerning its practice as described in the detaileddescription and examples, are hereby incorporated by reference in theirentirety.

125 24 base pairs nucleic acid single linear 1 ATCTCTACGG TGGTCCTAAATAGT 24 48 base pairs nucleic acid single linear 2 GGTGGAGCTC TAATACGACTCACTATAGAT TGACGGCGTA GTACACAC 48 15 base pairs nucleic acid singlelinear 3 AATTTCTGCC TCAGC 15 17 base pairs nucleic acid single linear 4TATGCAAAGT TACTGAC 17 18 base pairs nucleic acid single linear 5CTGTCATTAC TTCATGTC 18 15 base pairs nucleic acid single linear 6GCGTGGATCA CTTTC 15 19 base pairs nucleic acid single linear 7ATTGCGTGAT TTCGTCCGT 19 15 base pairs nucleic acid single linear 8TAAATTTGAG CTTTG 15 18 base pairs nucleic acid single linear 9GGCATATGGC ATTAGTTG 18 19 base pairs nucleic acid single linear 10CTGGCCATGG AAGGAAAGG 19 58 base pairs nucleic acid single linear 11CCCCTCGAGG GTTTTTTTTT TTTTTTTTTT TTGAAATGTT AAAAACAAAA TTTTGTTG 58 48base pairs nucleic acid single linear 12 TATATGGGCC CGATTTAGGTGACACTATAG ATTGACGGCG TAGTACAC 48 23 base pairs nucleic acid singlelinear 13 CTGGCAACCG GTAAGTACGA TAC 23 21 base pairs nucleic acid singlelinear 14 ATACTAGCCA CGGCCGGTAT C 21 21 base pairs nucleic acid singlelinear 15 TCCTCTTTCG ACGTGTCGAG C 21 21 base pairs nucleic acid singlelinear 16 ACCTTGGAGC GCAATGTCCT G 21 21 base pairs nucleic acid singlelinear 17 CCTTTTCAGG GGATCCGCCA C 21 21 base pairs nucleic acid singlelinear 18 GTGGCGGATC CCCTGAAAAG G 21 20 base pairs nucleic acid singlelinear 19 TGGGCCGTGT GGTCGTCATG 20 21 base pairs nucleic acid singlelinear 20 TGGGTCTTCA ACTCACCGGA C 21 22 base pairs nucleic acid singlelinear 21 CAATTCGACG TACGCCTCAC TC 22 22 base pairs nucleic acid singlelinear 22 GAGTGAGGCG TACGTCGAAT TG 22 52 base pairs nucleic acid singlelinear 23 TATATTCTAG ATTTTTTTTT TTTTTTTTTT TTTTTTTTTT TTTTTTGAAA TG 5235 base pairs nucleic acid single linear 24 ATATATCTAG AGCCATGGGCCACACACGGA GGCAG 35 35 base pairs nucleic acid single linear 25ATATAGGATC CCTGTTATAC AGGGCGTACA CTTTC 35 35 base pairs nucleic acidsingle linear 26 ATATACTCGA GACCATGATT GAACAAGATG GATTG 35 35 base pairsnucleic acid single linear 27 TATATAGCGG CCGCTCAGAA GAACTCGTCA AGAAG 3521 base pairs nucleic acid single linear 28 CTGTAGATGG TGACGGTGTC G 2122 base pairs nucleic acid single linear 29 GAAGTGCCAG AACAGCCTAC CG 2234 base pairs nucleic acid single linear 30 TATATCTCGA GGGTGGTGTTGTAGTATTAG TCAG 34 41 base pairs nucleic acid single linear 31TATATATATA TGCGGCCGCC GCTACGCCCC AATGATCCGA C 41 65 base pairs nucleicacid single linear 32 CTATAGAGCT CGTTTAAACT TTTTTTTTTT TTTTTTTTTTTTTTTTTTTT TTTTTTTTTG 60 AAATG 65 12 base pairs nucleic acid singlelinear 33 TCGATCCTAG GA 12 11 base pairs nucleic acid single linear 34AGGATCCTAG T 11 20 base pairs nucleic acid single linear 35 CTCGATCCTAGGATCGAGGC 20 20 base pairs nucleic acid single linear 36 GAGCTAGGATCCTAGCTCCG 20 44 base pairs nucleic acid single linear 37 TATATATGAGCTCTAATAAA ATGAGGAAAT TGCATCGCAT TGTC 44 43 base pairs nucleic acidsingle linear 38 TATATGAATT CATAGAATGA CACCTACTCA GACAATGCGA TGC 43 56base pairs nucleic acid single linear 39 AAAAAAAAAA GGGTCGGCATGGCATCTCCA CCTCCTCGCG GTCCGACCTG GGCATC 56 69 base pairs nucleic acidsingle linear 40 TATATGAGCT CCTCCCTTAG CCATCCGAGT GGACGTGCGT CCTCCTTCGGATGCCCAGGT 60 CGGACCGCG 69 34 base pairs nucleic acid single linear 41TATATATAGA TCTTTGACAT TGATTATTGA CTAG 34 42 base pairs nucleic acidsingle linear 42 CCGTCAATAC GGTTCACTAA ACGAGCTCTG CTTATATAGA CC 42 38base pairs nucleic acid single linear 43 GCTCGTTTAG TGAACCGTATTGACGGCGTA GTACACAC 38 23 base pairs nucleic acid single linear 44CTGGCAACCG GTAAGTACGA TAC 23 22 base pairs nucleic acid single linear 45GGTAACAAGA TCTCGTGCCG TG 22 34 base pairs nucleic acid single linear 46CCTATGCGGC CGCGTGGAAC CTTTGTGGCT CCTC 34 31 base pairs nucleic acidsingle linear 47 CCTATTGGCC AGCAGACCAA TTTATGCCTA C 31 34 base pairsnucleic acid single linear 48 CCTATGCGGC CGCTAGACTG GACAGCCAAT GACG 3432 base pairs nucleic acid single linear 49 CCTATTGGCC AGCCAAGACATCATCCGGGC AG 32 39 base pairs nucleic acid single linear 50 TATATATCCGGAAAGCTCTA AGGTAAATAT AAAATTTTT 39 43 base pairs nucleic acid singlelinear 51 TATATAGGAT CCTAGGTTAG GTTGGAATCT AAAATACACA AAC 43 12 basepairs nucleic acid single linear 52 TCGATCCTAG GA 12 16 base pairsnucleic acid single linear 53 GCTCTTAATT AAGAGC 16 55 base pairs nucleicacid single linear 54 CTGTTTAAAC AGATCTTATC TCGAGTATGC GGCCGCTATGAATTCGTTTA AACGA 55 55 base pairs nucleic acid single linear 55TCGTTTAAAC GAATTCATAG CGGCCGCATA CTCGAGATAA GATCTGTTTA AACAG 55 37 basepairs nucleic acid single linear 56 ATATATCCGG AGTCCGGCCG CTTGGGTGGAGAGGCTA 37 36 base pairs nucleic acid single linear 57 ATATAGGATCCTCAGAAGAA CTCGTCAAGA AGGCGA 36 44 base pairs nucleic acid single linear58 TATATATGAG CTCTTACAAA TAAAGCAATA GCATCACAAA TTTC 44 36 base pairsnucleic acid single linear 59 TATATGAATT CGTTTGGACA AACCACAACT AGAATG 3635 base pairs nucleic acid single linear 60 TATATAGATC TAGTCTTATGCAATACTCTT GTAGT 35 44 base pairs nucleic acid single linear 61GGGATACTCA CCACTATATC TCGACGGTAT CGAGGTAGGG CACT 44 22 base pairsnucleic acid single linear 62 GATATAGTGG TGAGTATCCC CG 22 34 base pairsnucleic acid single linear 63 TATATGGATC CCGGAGATCC TTAATCTTCT CATG 3428 base pairs nucleic acid single linear 64 TATATATGCA TCCCCCCCCCCCCCAACG 28 43 base pairs nucleic acid single linear 65 CATGCGAAACGATCCTCATC CTTACAATCG TGGTTTTCAA AGG 43 25 base pairs nucleic acidsingle linear 66 GATGAGGATC GTTTCGCATG ATTGA 25 36 base pairs nucleicacid single linear 67 TATATATGCA TTCAGAAGAA CTCGTCAAGA AGGCGA 36 35 basepairs nucleic acid single linear 68 ATATACTCGA GACCACCACC ATGAATAGAGGATTC 35 42 base pairs nucleic acid single linear 69 TATATGCGGCCGCTATTACC ACTCTTCTGT CCCTTCCGGG GT 42 42 base pairs nucleic acid singlelinear 70 ATATACTCGA GAGCAATGTC CGCAGCACCA CTGGTCACGG CA 42 39 basepairs nucleic acid single linear 71 ATATAGGCGG CCGCTCATCT TCGTGTGCTAGTCAGCATC 39 34 base pairs nucleic acid single linear 72 ACACATTAATTAACGATGCC GCCGGAAGCG AGAA 34 34 base pairs nucleic acid single linear73 ACACATTAAT TAAGTATTGG CCCCAATGGG GTCT 34 26 base pairs nucleic acidsingle linear 74 CCACGAATTC GGTCCTAAAT AGATGC 26 25 base pairs nucleicacid single linear 75 CCACAAGCTT CCGGGCGAGG CCGCC 25 23 base pairsnucleic acid single linear 76 CCACGGATCC CGGCGTTCCG TCC 23 25 base pairsnucleic acid single linear 77 CCACAAGCTT GTGCACTGGG ATCTG 25 25 basepairs nucleic acid single linear 78 CCACGGATCC GTGCACATGA AGTCC 25 30base pairs nucleic acid single linear 79 CCACAAGCTT CCGGAGTTACCCGAGTGACC 30 26 base pairs nucleic acid single linear 80 CCACCTTAAGCGTCGGCTTT TTCTTC 26 25 base pairs nucleic acid single linear 81CCACCTTAAG AGAAGAGAAA GAATG 25 24 base pairs nucleic acid single linear82 CCACAAGCTT GGACCACCGT AGAG 24 19 base pairs nucleic acid singlelinear 83 CCGCGTGGCG GATCCCCTG 19 24 base pairs nucleic acid singlelinear 84 CCACGGATCC GGAAGGGACA GAAG 24 16 base pairs nucleic acidsingle linear 85 CACGGTCCTG AGGTGC 16 42 base pairs nucleic acid singlelinear 86 TATATATATC TCGAGACCGC CAAGATGTTC CCGTTCCAGC CA 42 38 basepairs nucleic acid single linear 87 TATATATATG CGGCCGCTCA ATTATGTTTCTGGTTGGT 38 42 base pairs nucleic acid single linear 88 TATATAGATCTGGCGCGCCT TTACCACTCC TATCAGTGAT AG 42 35 base pairs nucleic acid singlelinear 89 TACGCCGTCA ATACGGTTCA CTAAACGAGC TCTGC 35 35 base pairsnucleic acid single linear 90 TAGTGAACCG TATTGACGGC GTAGTACACA CTATT 3522 base pairs nucleic acid single linear 91 CGTTGAGCAT AACCGAATCT AC 2242 base pairs nucleic acid single linear 92 ATATAGAGCT CTTAATTAATCTTTGTGAAG GAACCTTACT TC 42 35 base pairs nucleic acid single linear 93ATATAGAGCT CAGGCGTTGA AAAGATTAGC GACCG 35 46 base pairs nucleic acidsingle linear 94 TATATATTAA TTAAATAGAA TGACACCTAC TCAGACAATG CGATGC 4640 base pairs nucleic acid single linear 95 ATATACTCGA GTAGCAATGGTGAAACCAGT AACGTTATAC 40 45 base pairs nucleic acid single linear 96GCCCTTTCTC TTCTTTTTTG GCTGCCCGCT TTCCAGTCGG GAAAC 45 39 base pairsnucleic acid single linear 97 CCAAAAAAGA AGAGAAAGGG CGGTGGTGCT TTGTCTCCT39 38 base pairs nucleic acid single linear 98 ATATAGAGCT CTTAAACTGATGATTTGATT TCAAATGC 38 30 base pairs nucleic acid single linear 99CGCGCCGAAT TGTGAGCGCT CACAATTCGG 30 7 amino acids amino acid singlelinear 100 Pro Lys Lys Lys Lys Arg Lys 1 5 8000 base pairs nucleic acidsingle linear 101 ATTGACGGCG TAGTACACAC TATTGAATCA AACAGCCGAC CAATCGCACTACCATCACAA 60 TGGAGAAGCC AGTAGTAAAC GTAGACGTAG ACCCCCAGAG TCCGTTTGTCGTGCAACTGA 120 AAAAAAGCTT CCCGCAATTT GAGGTAGTAG CACAGCAGGT CACTCCAAATGACCATGCTA 180 ATGCCAGAGC ATTTTCGCAT CTGGCCAGTA AACTAATCGA GCTGGAGGTTCCTACCACAG 240 CGACGATCTT GGACATAGGC AGCGCACCGG CTCGTAGAAT GTTTTCCGAGCACCAGTATC 300 ATTGTGTCTG CCCCATGCGT AGTCCAGAAG ACCCGGACCG CATGATGAAATACGCCAGTA 360 AACTGGCGGA AAAAGCGTGC AAGATTACAA ACAAGAACTT GCATGAGAAGATTAAGGATC 420 TCCGGACCGT ACTTGATACG CCGGATGCTG AAACACCATC GCTCTGCTTTCACAACGATG 480 TTACCTGCAA CATGCGTGCC GAATATTCCG TCATGCAGGA CGTGTATATCAACGCTCCCG 540 GAACTATCTA TCATCAGGCT ATGAAAGGCG TGCGGACCCT GTACTGGATTGGCTTCGACA 600 CCACCCAGTT CATGTTCTCG GCTATGGCAG GTTCGTACCC TGCGTACAACACCAACTGGG 660 CCGACGAGAA AGTCCTTGAA GCGCGTAACA TCGGACTTTG CAGCACAAAGCTGAGTGAAG 720 GTAGGACAGG AAAATTGTCG ATAATGAGGA AGAAGGAGTT GAAGCCCGGGTCGCGGGTTT 780 ATTTCTCCGT AGGATCGACA CTTTATCCAG AACACAGAGC CAGCTTGCAGAGCTGGCATC 840 TTCCATCGGT GTTCCACTTG AATGGAAAGC AGTCGTACAC TTGCCGCTGTGATACAGTGG 900 TGAGTTGCGA AGGCTACGTA GTGAAGAAAA TCACCATCAG TCCCGGGATCACGGGAGAAA 960 CCGTGGGATA CGCGGTTACA CACAATAGCG AGGGCTTCTT GCTATGCAAAGTTACTGACA 1020 CAGTAAAAGG AGAACGGGTA TCGTTCCCTG TGTGCACGTA CATCCCGGCCACCATATGCG 1080 ATCAGATGAC TGGTATAATG GCCACGGATA TATCACCTGA CGATGCACAAAAACTTCTGG 1140 TTGGGCTCAA CCAGCGAATT GTCATTAACG GTAGGACTAA CAGGAACACCAACACCATGC 1200 AAAATTACCT TCTGCCGATC ATAGCACAAG GGTTCAGCAA ATGGGCTAAGGAGCGCAAGG 1260 ATGATCTTGA TAACGAGAAA ATGCTGGGTA CTAGAGAACG CAAGCTTACGTATGGCTGCT 1320 TGTGGGCGTT TCGCACTAAG AAAGTACATT CGTTTTATCG CCCACCTGGAACGCAGACCT 1380 GCGTAAAAGT CCCAGCCTCT TTTAGCGCTT TTCCCATGTC GTCCGTATGGACGACCTCTT 1440 TGCCCATGTC GCTGAGGCAG AAATTGAAAC TGGCATTGCA ACCAAAGAAGGAGGAAAAAC 1500 TGCTGCAGGT CTCGGAGGAA TTAGTCATGG AGGCCAAGGC TGCTTTTGAGGATGCTCAGG 1560 AGGAAGCCAG AGCGGAGAAG CTCCGAGAAG CACTTCCACC ATTAGTGGCAGACAAAGGCA 1620 TCGAGGCAGC CGCAGAAGTT GTCTGCGAAG TGGAGGGGCT CCAGGCGGACATCGGAGCAG 1680 CATTAGTTGA AACCCCGCGC GGTCACGTAA GGATAATACC TCAAGCAAATGACCGTATGA 1740 TCGGACAGTA TATCGTTGTC TCGCCAAACT CTGTACTGAA GAATGCCAAACTCGCACCAG 1800 CGCACCCGCT AGCAGATCAG GTTAAGATCA TAACACACTC CGGAAGATCAGGAAGGTACG 1860 CGGTCGAACC ATACGACGCT AAAGTACTGA TGCCAGCAGG AGGTGCCGTACCATGGCCAG 1920 AATTCCTAGC ACTGAGTGAG AGCGCCACGT TAGTGTACAA CGAAAGAGAGCTTGTGAACC 1980 GCAAACTATA CCACATTGCC ATGCATGGCC CCGCCAAGAA TACAGAAGAGGAGCAGTACA 2040 AGGTTACAAA GGCAGAGCTT GCAGAAACAG AGTACGTGTT TGACGTGGACAAGAAGCGTT 2100 GCGTTAAGAA GGAAGAAGCC TCAGGTCTGG TCCTCTCGGG AGAACTGACCAACCCTCCCT 2160 ATCATGAGCT AGCTCTGGAG GGACTGAAGA CCCGACCTGC GGTCCCGTACAAGGTCGAAA 2220 CAATAGGAGT GATAGGCACA CCGGGGTCGG GCAAGTCAGC TATTATCAAGTCAACTGTCA 2280 CGGCACGAGA TCTTGTTACC AGCGGAAAGA AAGAAAATTG TCGCGAAATTGAGGCCGACG 2340 TGCTAAGACT GAGGGGTATG CAGATTACGT CGAAGACAGT AGATTCGGTTATGCTCAACG 2400 GATGCCACAA AGCCGTAGAA GTGCTGTACG TTGACGAAGC GTTCGCGTGCCACGCAGGAG 2460 CACTACTTGC CTTGATTGCT ATCGTCAGGC CCCGCAAGAA GGTAGTACTATGCGGAGACC 2520 CCATGCAATG CGGATTCTTC AACATGATGC AACTAAAGGT ACATTTCAATCACCCTGAAA 2580 AAGACATATG CACCAAGACA TTCTACAAGT ATATCTCCCG GCGTTGCACACAGCCAGTTA 2640 CAGCTATTGT ATCGACACTG CATTACGATG GAAAGATGAA AACCACGAACCCGTGCAAGA 2700 AGAACATTGA AATCGATATT ACAGGGGCCA CAAAGCCGAA GCCAGGGGATATCATCCTGA 2760 CATGTTTCCG CGGGTGGGTT AAGCAATTGC AAATCGACTA TCCCGGACATGAAGTAATGA 2820 CAGCCGCGGC CTCACAAGGG CTAACCAGAA AAGGAGTGTA TGCCGTCCGGCAAAAAGTCA 2880 ATGAAAACCC ACTGTACGCG ATCACATCAG AGCATGTGAA CGTGTTGCTCACCCGCACTG 2940 AGGACAGGCT AGTGTGGAAA ACCTTGCAGG GCGACCCATG GATTAAGCAGCTCACTAACA 3000 TACCTAAAGG AAACTTTCAG GCTACTATAG AGGACTGGGA AGCTGAACACAAGGGAATAA 3060 TTGCTGCAAT AAACAGCCCC ACTCCCCGTG CCAATCCGTT CAGCTGCAAGACCAACGTTT 3120 GCTGGGCGAA AGCATTGGAA CCGATACTAG CCACGGCCGG TATCGTACTTACCGGTTGCC 3180 AGTGGAGCGA ACTGTTCCCA CAGTTTGCGG ATGACAAACC ACATTCGGCCATTTACGCCT 3240 TAGACGTAAT TTGCATTAAG TTTTTCGGCA TGGACTTGAC AAGCGGACTGTTTTCTAAAC 3300 AGAGCATCCC ACTAACGTAC CATCCCGCCG ATTCAGCGAG GCCGGTAGCTCATTGGGACA 3360 ACAGCCCAGG AACCCGCAAG TATGGGTACG ATCACGCCAT TGCCGCCGAACTCTCCCGTA 3420 GATTTCCGGT GTTCCAGCTA GCTGGGAAGG GCACACAACT TGATTTGCAGACGGGGAGAA 3480 CCAGAGTTAT CTCTGCACAG CATAACCTGG TCCCGGTGAA CCGCAATCTTCCTCACGCCT 3540 TAGTCCCCGA GTACAAGGAG AAGCAACCCG GCCCGGTCGA AAAATTCTTGAACCAGTTCA 3600 AACACCACTC AGTACTTGTG GTATCAGAGG AAAAAATTGA AGCTCCCCGTAAGAGAATCG 3660 AATGGATCGC CCCGATTGGC ATAGCCGGTG CAGATAAGAA CTACAACCTGGCTTTCGGGT 3720 TTCCGCCGCA GGCACGGTAC GACCTGGTGT TCATCAACAT TGGAACTAAATACAGAAACC 3780 ACCACTTTCA GCAGTGCGAA GACCATGCGG CGACCTTAAA AACCCTTTCGCGTTCGGCCC 3840 TGAATTGCCT TAACTCAGGA GGCACTCTCG TGGTGAAGTC CTATGGCTACGCCGACCGCA 3900 ACAGTGAGGA CGTAGTCACC GCTCTTGCCA GAAAGTTTGT CAGGGTGTCTGCAGCGAGAC 3960 CAGATTGTGT CTCAAGCAAT ACAGAAATGT ACCTGATTTT CCGACAACTAGACAACAGCC 4020 GTACACGGCA ATTCACCCCG CACCATCTGA ATTGCGTGAT TTCGTCCGTGTATGAGGGTA 4080 CAAGAGATGG AGTTGGAGCC GCGCCGTCAT ACCGCACCAA AAGGGAGAATATTGCTGACT 4140 GTCAAGAGGA AGCAGTTGTC AACGCAGCCA ATCCGCTGGG TAGACCAGGCGAAGGAGTCT 4200 GCCGTGCCAT CTATAAACGT TGGCCGACCA GTTTTACCGA TTCAGCCACGGAGACAGGCA 4260 CCGCAAGAAT GACTGTGTGC CTAGGAAAGA AAGTGATCCA CGCGGTCGGCCCTGATTTCC 4320 GGAAGCACCC AGAAGCAGTA GCCTTGAAAT TGCTACAAAA CGCCTACCATGCAGTGGCAG 4380 ACTTAGTAAA TGAACATAAC ATCAAGTCTG TCGCCATTCC ACTGCTATCTACAGGCATTT 4440 ACGCAGCCGG AAAAGACCGC CTTGAAGTAT CACTTAACTG CTTGACAACCGCGCTAGACA 4500 GAACTGACGC GGACGTAACC ATCTATTGCC TGGATAAGAA GTGGAAGGAAAGAATCGACG 4560 CGGCACTCCA ACTTAAGGAG TCTGTAACAG AGCTGAAGGA TGAAGATATGGAGATCGACG 4620 ATGAGTTAGT ATGGATCCAT CCAGACAGTT GCTTGAAGGG AAGAAAGGGATTCAGTACTA 4680 CAAAAGGAAA ATTGTATTCG TACTTCGAAG GCACCAAATT CCATCAAGCAGCAAAAGACA 4740 TGGCGGAGAT AAAGGTCCTG TTCCCTAATG ACCAGGAAAG TAATGAACAACTGTGTGCCT 4800 ACATATTGGG TGAGACCATG GAAGCAATCC GCGAAAAGTG CCCGGTCGACCATAACCCGT 4860 CGTTTAGCCC GCCCAAAACG TTGCCGTGCC TTTGCATGTA TGCCATGACGCCAGAAAGGG 4920 TCCACAGACT TAGAAGCAAT AACGTCAAAG AAGTTACAGT ATGCTCCTCCACCCCCCTTC 4980 CTAAGCACAA AATTAAGAAT GTTCAGAAGG TTCAGTGCAC GAAAGTAGTCCTGTTTAATC 5040 CGCACACTCC CGCATTCGTT CCCGCCCGTA AGTACATAGA AGTGCCAGAACAGCCTACCG 5100 CTCCTCCTGC ACAGGCCGAG GAGGCCCCCG AAGTTGTAGC GACACCGTCACCATCTACAG 5160 CTGATAACAC CTCGCTTGAT GTCACAGACA TCTCACTGGA TATGGATGACAGTAGCGAAG 5220 GCTCACTTTT TTCGAGCTTT AGCGGATCGG ACAACTCTAT TACTAGTATGGACAGTTGGT 5280 CGTCAGGACC TAGTTCACTA GAGATAGTAG ACCGAAGGCA GGTGGTGGTGGCTGACGTTC 5340 ATGCCGTCCA AGAGCCTGCC CCTATTCCAC CGCCAAGGCT AAAGAAGATGGCCCGCCTGG 5400 CAGCGGCAAG AAAAGAGCCC ACTCCACCGG CAAGCAATAG CTCTGAGTCCCTCCACCTCT 5460 CTTTTGGTGG GGTATCCATG TCCCTCGGAT CAATTTTCGA CGGAGAGACGGCCCGCCAGG 5520 CAGCGGTACA ACCCCTGGCA ACAGGCCCCA CGGATGTGCC TATGTCTTTCGGATCGTTTT 5580 CCGACGGAGA GATTGATGAG CTGAGCCGCA GAGTAACTGA GTCCGAACCCGTCCTGTTTG 5640 GATCATTTGA ACCGGGCGAA GTGAACTCAA TTATATCGTC CCGATCAGCCGTATCTTTTC 5700 CTCTACGCAA GCAGAGACGT AGACGCAGGA GCAGGAGGAC TGAATACTGACTAACCGGGG 5760 TAGGTGGGTA CATATTTTCG ACGGACACAG GCCCTGGGCA CTTGCAAAAGAAGTCCGTTC 5820 TGCAGAACCA GCTTACAGAA CCGACCTTGG AGCACAATGT CCTGGAAAGAATTCATGCCC 5880 CGGTGCTCGA CACGTCGAAA GAGGAACAAC TCAAACTCAG GTACCAGATGATGCCCACCG 5940 AAGCCAACAA AAGTAGGTAC CAGTCTCGTA AAGTAGAAAA TCAGAAAGCCATAACCACTG 6000 AGCGACTACT GTCAGGACTA CGACTGTATA ACTCTGCCAC AGATCAGCCAGAATGCTATA 6060 AGATCACCTA TCCGAAACCA TTGTACTCCA GTAGCGTACC GGCGAACTACTCCGATCCAC 6120 AGTTCGCTGT AGCTGTCTGT AACAACTATC TGCATGAGAA CTATCCGACAGTAGCATCTT 6180 ATCAGATTAC TGACGAGTAC GATGCTTACT TGGATATGGT AGACGGGACAGTCGCCTGCC 6240 TGGATACTGC AACCTTCTGC CCCGCTAAGC TTAGAAGTTA CCCGAAAAAACATGAGTATA 6300 GAGCCCCGAA TATCCGCAGT GCGGTTCCAT CAGCGATGCA GAACACGCTACAAAATGTGC 6360 TCATTGCCGC AACTAAAAGA AATTGCAACG TCACGCAGAT GCGTGAACTGCCAACACTGG 6420 ACTCAGCGAC ATTCAATGTC GAATGCTTTC GAAAATATGC ATGTAATGACGAGTATTGGG 6480 AGGAGTTCGC TCGGAAGCCA ATTAGGATTA CCACTGAGTT TGTCACCGCATATGTAGCTA 6540 GACTGAAAGG CCCTAAGGCC GCCGCACTAT TTGCAAAGAC GTATAATTTGGTCCCATTGC 6600 AAGAAGTGCC TATGGATAGA TTCGTCATGG ACATGAAAAG AGACGTGAAAGTTACACCAG 6660 GCACGAAACA CACAGAAGAA AGACCGAAAG TACAAGTGAT ACAAGCCGCAGAACCCCTGG 6720 CGACTGCTTA CTTATGCGGG ATTCACCGGG AATTAGTGCG TAGGCTTACGGCCGTCTTGC 6780 TTCCAAACAT TCACACGCTT TTTGACATGT CGGCGGAGGA TTTTGATGCAATCATAGCAG 6840 AACACTTCAA GCAAGGCGAC CCGGTACTGG AGACGGATAT CGCATCATTCGACAAAAGCC 6900 AAGACGACGC TATGGCGTTA ACCGGTCTGA TGATCTTGGA GGACCTGGGTGTGGATCAAC 6960 CACTACTCGA CTTGATCGAG TGCGCCTTTG GAGAAATATC ATCCACCCATCTACCTACGG 7020 GTACTCGTTT TAAATTCGGG GCGATGATGA AATCCGGAAT GTTCCTCACACTTTTTGTCA 7080 ACACAGTTTT GAATGTCGTT ATCGCCAGCA GAGTACTAGA AGAGCGGCTTAAAACGTCCA 7140 GATGTGCAGC GTTCATTGGC GACGACAACA TCATACATGG AGTAGTATCTGACAAAGAAA 7200 TGGCTGAGAG GTGCGCCACC TGGCTCAACA TGGAGGTTAA GATCATCGACGCAGTCATCG 7260 GTGAGAGACC ACCTTACTTC TGCGGCGGAT TTATCTTGCA AGATTCGGTTACTTCCACAG 7320 CGTGCCGCGT GGCGGATCCC CTGAAAAGGC TGTTTAAGTT GGGTAAACCGCTCCCAGCCG 7380 ACGACGAGCA AGACGAAGAC AGAAGACGCG CTCTGCTAGA TGAAACAAAGGCGTGGTTTA 7440 GAGTAGGTAT AACAGGCACT TTAGCAGTGG CCGTGACGAC CCGGTATGAGGTAGACAATA 7500 TTACACCTGT CCTACTGGCA TTGAGAACTT TTGCCCAGAG CAAAAGAGCATTCCAAGCCA 7560 TCAGAGGGGA AATAAAGCAT CTCTACGGTG GTCCTAAATA GTCAGCATAGTTCATTTCAT 7620 CTGACTAATA CTACAACACC ACCACCATGA ATAGAGGATT CTTTAACATGCTCGGCCGCC 7680 GCCCCTTCCC GGCCCCCACT GCCATGTGGA GGCCGCGGAG AAGGAGGCAGGCGGCCCCGA 7740 TGCCTGCCCG CAACGGGCTG GCTTCTCAAA TCCAGCAACT GACCACAGCCGTCAGTGCCC 7800 TAGTCATTGG ACAGGCAACT AGACCTCAAC CCCCATGTCC ACGCCCGCCACCGCGCCAGA 7860 AGAAGCAGGC GCCCAAGCAA CCACCGAAGC CGAAGAAACC AAAAACGCAGGAGAAGAAGA 7920 AGAAGCAACC TGCAAAACCC AAACCCGGAA AGAGACAGCG CATGGCACTTAAGTTGGAGG 7980 CCGACAGATT GTTCGACGTC 8000 8000 base pairs nucleic acidsingle linear 102 ATTGACGGCG TAGTACACAC TATTGAATCA AACAGCCGAC CAATTGCACTACCATCACAA 60 TGGAGAAGCC AGTAGTAAAC GTAGACGTAG ACCCCCAGAG TCCGTTTGTCGTGCAACTGC 120 AAAAAAGCTT CCCGCAATTT GAGGTAGTAG CACAGCAGGT CACTCCAAATGACCATGCTA 180 ATGCCAGAGC ATTTTCGCAT CTGGCCAGTA AACTAATCGA GCTGGAGGTTCCTACCACAG 240 CGACGATCTT GGACATAGGC AGCGCACCGG CTCGTAGAAT GTTTTCCGAGCACCAGTATC 300 ATTGTGTCTG CCCCATGCGT AGTCCAGAAG ACCCGGACCG CATGATGAAATACGCCAGTA 360 AACTGGCGGA AAAAGCGTGC AAGATTACAA ACAAGAACTT GCATGAGAAGATTAAGGATC 420 TCCGGACCGT ACTTGATACG CCGGATGCTG AAACACCATC GCTCTGCTTTCACAACGATG 480 TTACCTGCAA CATGCGTGCC GAATATTCCG TCATGCAGGA CGTGTATATCAACGCTCCCG 540 GAACTATCTA TCATCAGGCT ATGAAAGGCG TGCGGACCCT GTACTGGATTGGCTTCGACA 600 CCACCCAGTT CATGTTCTCG GCTATGGCAG GTTCGTACCC TGCGTACAACACCAACTGGG 660 CCGACGAGAA AGTCCTTGAA GCGCGTAACA TCGGACTTTG CAGCACAAAGCTGAGTGAAG 720 GTAGGACAGG AAAATTGTCG ATAATGAGGA AGAAGGAGTT GAAGCCCGGGTCGCGGGTTT 780 ATTTCTCCGT AGGATCGACA CTTTATCCAG AACACAGAGC CAGCTTGCAGAGCTGGCATC 840 TTCCATCGGT GTTCCACTTG AATGGAAAGC AGTCGTACAC TTGCCGCTGTGATACAGTGG 900 TGAGTTGCGA AGGCTACGTA GTGAAGAAAA TCACCATCAG TCCCGGGATCACGGGAGAAA 960 CCGTGGGATA CGCGGTTACA CACAATAGCG AGGGCTTCTT GCTATGCAAAGTTACTGACA 1020 CAGTAAAAGG AGAACGGGTA TCGTTCCCTG TGTGCACGTA CATCCCGGCCACCATATGCG 1080 ATCAGATGAC TGGTATAATG GCCACGGATA TATCACCTGA CGATGCACAAAAACTTCTGG 1140 TTGGGCTCAA CCAGCGAATT GTCATTAACG GTAGGACTAA CAGGAACACCAACACCATGC 1200 AAAATTACCT TCTGCCGATC ATAGCACAAG GGTTCAGCAA ATGGGCTAAGGAGCGCAAGG 1260 ATGATCTTGA TAACGAGAAA ATGCTGGGTA CTAGAGAACG CAAGCTTACGTATGGCTGCT 1320 TGTGGGCGTT TCGCACTAAG AAAGTACATT CGTTTTATCG CCCACCTGGAACGCAGACCT 1380 GCGTAAAAGT CCCAGCCTCT TTTAGCGCTT TTCCCATGTC GTCCGTATGGACGACCTCTT 1440 TGCCCATGTC GCTGAGGCAG AAATTGAAAC TGGCATTGCA ACCAAAGAAGGAGGAAAAAC 1500 TGCTGCAGGT CTCGGAGGAA TTAGTCATGG AGGCCAAGGC TGCTTTTGAGGATGCTCAGG 1560 AGGAAGCCAG AGCGGAGAAG CTCCGAGAAG CACTTCCACC ATTAGTGGCAGACAAAGGCA 1620 TCGAGGCAGC CGCAGAAGTT GTCTGCGAAG TGGAGGGGCT CCAGGCGGACATCGGAGCAG 1680 CATTAGTTGA AACCCCGCGC GGTCACGTAA GGATAATACC TCAAGCAAATGACCGTATGA 1740 TCGGACAGTA TATCGTTGTC TCGCCAAACT CTGTGCTGAA GAATGCCAAACTCGCACCAG 1800 CGCACCCGCT AGCAGATCAG GTTAAGATCA TAACACACTC CGGAAGATCAGGAAGGTACG 1860 CGGTCGAACC ATACGACGCT AAAGTACTGA TGCCAGCAGG AGGTGCCGTACCATGGCCAG 1920 AATTCCTAGC ACTGAGTGAG AGCGCCACGT TAGTGTACAA CGAAAGAGAGTTTGTGAACC 1980 GCAAACTATA CCACATTGCC ATGCATGGCC CCGCCAAGAA TACAGAAGAGGAGCAGTACA 2040 AGGTTACAAA GGCAGAGCTT GCAGAAACAG AGTACGTGTT TGACGTGGACAAGAAGCGTT 2100 GCGTTAAGAA GGAAGAAGCC TCAGGTCTGG TCCTCTCGGG AGAACTGACCAACCCTCCCT 2160 ATCATGAGCT AGCTCTGGAG GGACTGAAGA CCCGACCTGC GGTCCCGTACAAGGTCGAAA 2220 CAATAGGAGT GATAGGCACA CCGGGGTCGG GCAAGTCAGC TATTATCAAGTCAACTGTCA 2280 CGGCACGAGA TCTTGTTACC AGCGGAAAGA AAGAAAATTG TCGCGAAATTGAGGCCGACG 2340 TGCTAAGACT GAGGGGTATG CAGATTACGT CGAAGACAGT AGATTCGGTTATGCTCAACG 2400 GATGCCACAA AGCCGTAGAA GTGCTGTACG TTGACGAAGC GTTCGCGTGCCACGCAGGAG 2460 CACTACTTGC CTTGATTGCT ATCGTCAGGC CCCGCAAGAA GGTAGTACTATGCGGAGACC 2520 CCATGCAATG CGGATTCTTC AACATGATGC AACTAAAGGT ACATTTCAATCACCCTGAAA 2580 AAGACATATG CACCAAGACA TTCTACAAGT ATATCTCCCG GCGTTGCACACAGCCAGTTA 2640 CAGCTATTGT ATCGACACTG CATTACGATG GAAAGATGAA AACCACGAACCCGTGCAAGA 2700 AGAACATTGA AATCGATATT ACAGGGGCCA CAAAGCCGAA GCCAGGGGATATCATCCTGA 2760 CATGTTTCCG CGGGTGGGTT AAGCAATTGC AAATCGACTA TCCCGGACATGAAGTAATGA 2820 CAGCCGCGGC CTCACAAGGG CTAACCAGAA AAGGAGTGTA TGCCGTCCGGCAAAAAGTCA 2880 ATGAAAACCC ACTGTACGCG ATCACATCAG AGCATGTGAA CGTGTTGCTCACCCGCACTG 2940 AGGACAGGCT AGTGTGGAAA ACCTTGCAGG GCGACCCATG GATTAAGCAGCCCACTAACA 3000 TACCTAAAGG AAACTTTCAG GCTACTATAG AGGACTGGGA AGCTGAACACAAGGGAATAA 3060 TTGCTGCAAT AAACAGCCCC ACTCCCCGTG CCAATCCGTT CAGCTGCAAGACCAACGTTT 3120 GCTGGGCGAA AGCATTGGAA CCGATACTAG CCACGGCCGG TATCGTACTTACCGGTTGCC 3180 AGTGGAGCGA ACTGTTCCCA CAGTTTGCGG ATGACAAACC ACATTCGGCCATTTACGCCT 3240 TAGACGTAAT TTGCATTAAG TTTTTCGGCA TGGACTTGAC AAGCGGACTGTTTTCTAAAC 3300 AGAGCATCCC ACTAACGTAC CATCCCGCCG ATTCAGCGAG GCCGGTAGCTCATTGGGACA 3360 ACAGCCCAGG AACCCGCAAG TATGGGTACG ATCACGCCAT TGCCGCCGAACTCTCCCGTA 3420 GATTTCCGGT GTTCCAGCTA GCTGGGAAGG GCACACAACT TGATTTGCAGACGGGGAGAA 3480 CCAGAGTTAT CTCTGCACAG CATAACCTGG TCCCGGTGAA CCGCAATCTTCCTCACGCCT 3540 TAGTCCCCGA GTACAAGGAG AAGCAACCCG GCCCGGTCAA AAAATTCTTGAACCAGTTCA 3600 AACACCACTC AGTACTTGTG GTATCAGAGG AAAAAATTGA AGCTCCCCGTAAGAGAATCG 3660 AATGGATCGC CCCGATTGGC ATAGCCGGTG CAGATAAGAA CTACAACCTGGCTTTCGGGT 3720 TTCCGCCGCA GGCACGGTAC GACCTGGTGT TCATCAACAT TGGAACTAAATACAGAAACC 3780 ACCACTTTCA GCAGTGCGAA GACCATGCGG CGACCTTAAA AACCCTTTCGCGTTCGGCCC 3840 TGAATTGCCT TAACCCAGGA GGCACCCTCG TGGTGAAGTC CTATGGCTACGCCGACCGCA 3900 ACAGTGAGGA CGTAGTCACC GCTCTTGCCA GAAAGTTTGT CAGGGTGTCTGCAGCGAGAC 3960 CAGATTGTGT CTCAAGCAAT ACAGAAATGT ACCTGATTTT CCGACAACTAGACAACAGCC 4020 GTACACGGCA ATTCACCCCG CACCATCTGA ATTGCGTGAT TTCGTCCGTGTATGAGGGTA 4080 CAAGAGATGG AGTTGGAGCC GCGCCGTCAT ACCGCACCAA AAGGGAGAATATTGCTGACT 4140 GTCAAGAGGA AGCAGTTGTC AACGCAGCCA ATCCGCTGGG TAGACCAGGCGAAGGAGTCT 4200 GCCGTGCCAT CTATAAACGT TGGCCGACCA GTTTTACCGA TTCAGCCACGGAGACAGGCA 4260 CCGCAAGAAT GACTGTGTGC CTAGGAAAGA AAGTGATCCA CGCGGTCGGCCCTGATTTCC 4320 GGAAGCACCC AGAAGCAGAA GCCTTGAAAT TGCTACAAAA CGCCTACCATGCAGTGGCAG 4380 ACTTAGTAAA TGAACATAAC ATCAAGTCTG TCGCCATTCC ACTGCTATCTACAGGCATTT 4440 ACGCAGCCGG AAAAGACCGC CTTGAAGTAT CACTTAACTG CTTGACAACCGCGCTAGACA 4500 GAACTGACGC GGACGTAACC ATCTATTGCC TGGATAAGAA GTGGAAGGAAAGAATCGACG 4560 CGGCACTCCA ACTTAAGGAG TCTGTAACAG AGCTGAAGGA TGAAGATATGGAGATCGACG 4620 ATGAGTTAGT ATGGATCCAT CCAGACAGTT GCTTGAAGGG AAGAAAGGGATTCAGTACTA 4680 CAAAAGGAAA ATTGTATTCG TACTTCGAAG GCACCAAATT CCATCAAGCAGCAAAAGACA 4740 TGGCGGAGAT AAAGGTCCTG TTCCCTAATG ACCAGGAAAG TAATGAACAACTGTGTGCCT 4800 ACATATTGGG TGAGACCATG GAAGCAATCC GCGAAAAGTG CCCGGTCGACCATAACCCGT 4860 CGTCTAGCCC GCCCAAAACG TTGCCGTGCC TTTGCATGTA TGCCATGACGCCAGAAAGGG 4920 TCCACAGACT TAGAAGCAAT AACGTCAAAG AAGTTACAGT ATGCTCCTCCACCCCCCTTC 4980 CTAAGCACAA AATTAAGAAT GTTCAGAAGG TTCAGTGCAC GAAAGTAGTCCTGTTTAATC 5040 CGCACACTCC CGCATTCGTT CCCGCCCGTA AGTACATAGA AGTGCCAGAACAGCCTACCG 5100 CTCCTCCTGC ACAGGCCGAG GAGGCCCCCG AAGTTGTAGC GACACCGTCACCATCTACAG 5160 CTGATAACAC CTCGCTTGAT GTCACAGACA TCTCACTGGA TATGGATGACAGTAGCGAAG 5220 GCTCACTTTT TTCGAGCTTT AGCGGATCGG ACAACTCTAT TACTAGTATGGACAGTTGGT 5280 CGTCAGGACC TAGTTCACTA GAGATAGTAG ACCGAAGGCA GGTGGTGGTGGCTGACGTTC 5340 ATGCCGTCCA AGAGCCTGCC CCTATTCCAC CGCCAAGGCT AAAGAAGATGGCCCGCCTGG 5400 CAGCGGCAAG AAAAGAGCCC ACTCCACCGG CAAGCAATAG CTCTGAGTCCCTCCACCTCT 5460 CTTTTGGTGG GGTATCCATG TCCCTCGGAT CAATTTTCGA CGGAGAGACGGCCCGCCAGG 5520 CAGCGGTACA ACCCCTGGCA ACAGGCCCCA CGGATGTGCC TATGTCTTTCGGATCGTTTT 5580 CCGACGGAGA GATTGATGAG CTGAGCCGCA GAGTAACTGA GTCCGAACCCGTCCTGTTTG 5640 GATCATTTGA ACCGGGCGAA GTGAACTCAA TTATATCGTC CCGATCAGCCGTATCTTTTC 5700 CACTACGCAA GCAGAGACGT AGACGCAGGA GCAGGAGGAC TGAATACTGACTAACCGGGG 5760 TAGGTGGGTA CATATTTTCG ACGGACACAG GCCCTGGGCA CTTGCAAAAGAAGTCCGTTC 5820 TGCAGAACCA GCTTACAGAA CCGACCTTGG AGCGCAATGT CCTGGAAAGAATTCATGCCC 5880 CGGTGCTCGA CACGTCGAAA GAGGAACAAC TCAAACTCAG GTACCAGATGATGCCCACCG 5940 AAGCCAACAA AAGTAGGTAC CAGTCTCGTA AAGTAGAAAA TCAGAAAGCCATAACCACTG 6000 AGCGACTACT GTCAGGACTA CGACTGTATA ACTCTGCCAC AGATCAGCCAGAATGCTATA 6060 AGATCACCTA TCCGAAACCA TTGTACTCCA GTAGCGTACC GGCGAACTACTCCGATCCAC 6120 AGTTCGCTGT AGCTGTCTGT AACAACTATC TGCATGAGAA CTATCCGACAGTAGCATCTT 6180 ATCAGATTAC TGACGAGTAC GATGCTTACT TGGATATGGT AGACGGGACAGTCGCCTGCC 6240 TGGATACTGC AACCTTCTGC CCCGCTAAGC TTAGAAGTTA CCCGAAAAAACATGAGTATA 6300 GAGCCCCGAA TATCCGCAGT GCGGTTCCAT CAGCGATGCA GAACACGCTACAAAATGTGC 6360 TCATTGCCGC AACTAAAAGA AATTGCAACG TCACGCAGAT GCGTGAACTGCCAACACTGG 6420 ACTCAGCGAC ATTCAATGTC GAATGCTTTC GAAAATATGC ATGTAATGACGAGTATTGGG 6480 AGGAGTTCGC TCGGAAGCCA ATTAGGATTA CCACTGAGTT TGTCACCGCATATGTAGCTA 6540 GACTGAAAGG CCCTAAGGCC GCCGCACTAT TTGCAAAGAC GTATAATTTGGTCCCATTGC 6600 AAGAAGTGCC TATGGATAGA TTCGTCATGG ACATGAAAAG AGACGTGAAAGTTACACCAG 6660 GCACGAAACA CACAGAAGAA AGACCGAAAG TACAAGTGAT ACAAGCCGCAGAACCCCTGG 6720 CGACTGCTTA CTTATGCGGG ATTCACCGGG AATTAGTGCG TAGGCTTACGGCCGTCTTGC 6780 TTCCAAACAT TCACACGCTT TTTGACATGT CGGCGGAGGA TTTTGATGCAATCATAGCAG 6840 AACACTTCAA GCAAGGCGAC CCGGTACTGG AGACGGATAT CGCATCATTCGACAAAAGCC 6900 AAGACGACGC TATGGCGTTA ACCGGTCTGA TGATCTTGGA GGACCTGGGTGTGGATCAAC 6960 CACTACTCGA CTTGATCGAG TGCGCCTTTG GAGAAATATC ATCCACCCATCTACCTACGG 7020 GTACTCGTTT TAAATTCGGG GCGATGATGA AATCCGGAAT GTTCCTCACACTTTTTGTCA 7080 ACACAGTTTT GAATGTCGTT ATCGCCAGCA GAGTACTAGA AGAGCGGCTTAAAACGTCCA 7140 GATGTGCAGC GTTCATTGGC GACGACAACA TCATACATGG AGTAGTATCTGACAAAGAAA 7200 TGGCTGAGAG GTGCGCCACC TGGCTCAACA TGGAGGTTAA GATCATCGACGCAGTCATCG 7260 GTGAGAGACC ACCTTACTTC TGCGGCGGAT TTATCTTGCA AGATTCGGTTACTTCCACAG 7320 CGTGCCGCGT GGCGGATCCC CTGAAAAGGC TGTTTAAGTT GGGTAAACCGCTCCCAGCCG 7380 ACGACGAGCA AGACGAAGAC AGAAGACGCG CTCTGCTAGA TGAAACAAAGGCGTGGTTTA 7440 GAGTAGGTAT AACAGGCACT TTAGCAGTGG CCGTGACGAC CCGGTATGAGGTAGACAATA 7500 TTACACCTGT CCTACTGGCA TTGAGAACTT TTGCCCAGAG CAAAAGAGCATTCCAAGCCA 7560 TCAGAGGGGA AATAAAGCAT CTCTACGGTG GTCCTAAATA GTCAGCATAGTACATTTCAT 7620 CTGACTAATA CTACAACACC ACCACCATGA ATAGAGGATT CTTTAACATGCTCGGCCGCC 7680 GCCCCTTCCC GGCCCCCACT GCCATGTGGA GGCCGCGGAG AAGGAGGCAGGCGGCCCCGA 7740 TGCCTGCCCG CAACGGGCTG GCTTCTCAAA TCCAGCAACT GACCACAGCCGTCAGTGCCC 7800 TAGTCATTGG ACAGGCAACT AGACCTCAAC CCCCACGTCC ACGCCCGCCACCGCGCCAGA 7860 AGAAGCAGGC GCCCAAGCAA CCACCGAAGC CGAAGAAACC AAAAACGCAGGAGAAGAAGA 7920 AGAAGCAACC TGCAAAACCC AAACCCGGAA AGAGACAGCG CATGGCACTTAAGTTGGAGG 7980 CCGACAGATT GTTCGACGTC 8000 11740 base pairs nucleic acidsingle linear 103 ATTGACGGCG TAGTACACAC TATTGAATCA AACAGCCGAC CAATTGCACTACCATCACAA 60 TGGAGAAGCC AGTAGTAAAC GTAGACGTAG ACCCCCAGAG TCCGTTTGTCGTGCAACTGC 120 AAAAAAGCTT CCCGCAATTT GAGGTAGTAG CACAGCAGGT CACTCCAAATGACCATGCTA 180 ATGCCAGAGC ATTTTCGCAT CTGGCCAGTA AACTAATCGA GCTGGAGGTTCCTACCACAG 240 CGACGATCTT GGACATAGGC AGCGCACCGG CTCGTAGAAT GTTTTCCGAGCACCAGTATC 300 ATTGTGTCTG CCCCATGCGT AGTCCAGAAG ACCCGGACCG CATGATGAAATACGCCAGTA 360 AACTGGCGGA AAAAGCGTGC AAGATTACAA ACAAGAACTT GCATGAGAAGATTAAGGATC 420 TCCGGACCGT ACTTGATACG CCGGATGCTG AAACACCATC GCTCTGCTTTCACAACGATG 480 TTACCTGCAA CATGCGTGCC GAATATTCCG TCATGCAGGA CGTGTATATCAACGCTCCCG 540 GAACTATCTA TCATCAGGCT ATGAAAGGCG TGCGGACCCT GTACTGGATTGGCTTCGACA 600 CCACCCAGTT CATGTTCTCG GCTATGGCAG GTTCGTACCC TGCGTACAACACCAACTGGG 660 CCGACGAGAA AGTCCTTGAA GCGCGTAACA TCGGACTTTG CAGCACAAAGCTGAGTGAAG 720 GTAGGACAGG AAAATTGTCG ATAATGAGGA AGAAGGAGTT GAAGCCCGGGTCGCGGGTTT 780 ATTTCTCCGT AGGATCGACA CTTTATCCAG AACACAGAGC CAGCTTGCAGAGCTGGCATC 840 TTCCATCGGT GTTCCACTTG AATGGAAAGC AGTCGTACAC TTGCCGCTGTGATACAGTGG 900 TGAGTTGCGA AGGCTACGTA GTGAAGAAAA TCACCATCAG TCCCGGGATCACGGGAGAAA 960 CCGTGGGATA CGCGGTTACA CACAATAGCG AGGGCTTCTT GCTATGCAAAGTTACTGACA 1020 CAGTAAAAGG AGAACGGGTA TCGTTCCCTG TGTGCACGTA CATCCCGGCCACCATATGCG 1080 ATCAGATGAC TGGTATAATG GCCACGGATA TATCACCTGA CGATGCACAAAAACTTCTGG 1140 TTGGGCTCAA CCAGCGAATT GTCATTAACG GTAGGACTAA CAGGAACACCAACACCATGC 1200 AAAATTACCT TCTGCCGATC ATAGCACAAG GGTTCAGCAA ATGGGCTAAGGAGCGCAAGG 1260 ATGATCTTGA TAACGAGAAA ATGCTGGGTA CTAGAGAACG CAAGCTTACGTATGGCTGCT 1320 TGTGGGCGTT TCGCACTAAG AAAGTACATT CGTTTTATCG CCCACCTGGAACGCAGACCT 1380 GCGTAAAAGT CCCAGCCTCT TTTAGCGCTT TTCCCATGTC GTCCGTATGGACGACCTCTT 1440 TGCCCATGTC GCTGAGGCAG AAATTGAAAC TGGCATTGCA ACCAAAGAAGGAGGAAAAAC 1500 TGCTGCAGGT CTCGGAGGAA TTAGTCATGG AGGCCAAGGC TGCTTTTGAGGATGCTCAGG 1560 AGGAAGCCAG AGCGGAGAAG CTCCGAGAAG CACTTCCACC ATTAGTGGCAGACAAAGGCA 1620 TCGAGGCAGC CGCAGAAGTT GTCTGCGAAG TGGAGGGGCT CCAGGCGGACATCGGAGCAG 1680 CATTAGTTGA AACCCCGCGC GGTCACGTAA GGATAATACC TCAAGCAAATGACCGTATGA 1740 TCGGACAGTA TATCGTTGTC TCGCCAAACT CTGTGCTGAA GAATGCCAAACTCGCACCAG 1800 CGCACCCGCT AGCAGATCAG GTTAAGATCA TAACACACTC CGGAAGATCAGGAAGGTACG 1860 CGGTCGAACC ATACGACGCT AAAGTACTGA TGCCAGCAGG AGGTGCCGTACCATGGCCAG 1920 AATTCCTAGC ACTGAGTGAG AGCGCCACGT TAGTGTACAA CGAAAGAGAGTTTGTGAACC 1980 GCAAACTATA CCACATTGCC ATGCATGGCC CCGCCAAGAA TACAGAAGAGGAGCAGTACA 2040 AGGTTACAAA GGCAGAGCTT GCAGAAACAG AGTACGTGTT TGACGTGGACAAGAAGCGTT 2100 GCGTTAAGAA GGAAGAAGCC TCAGGTCTGG TCCTCTCGGG AGAACTGACCAACCCTCCCT 2160 ATCATGAGCT AGCTCTGGAG GGACTGAAGA CCCGACCTGC GGTCCCGTACAAGGTCGAAA 2220 CAATAGGAGT GATAGGCACA CCGGGGTCGG GCAAGTCAGC TATTATCAAGTCAACTGTCA 2280 CGGCACGAGA TCTTGTTACC AGCGGAAAGA AAGAAAATTG TCGCGAAATTGAGGCCGACG 2340 TGCTAAGACT GAGGGGTATG CAGATTACGT CGAAGACAGT AGATTCGGTTATGCTCAACG 2400 GATGCCACAA AGCCGTAGAA GTGCTGTACG TTGACGAAGC GTTCGCGTGCCACGCAGGAG 2460 CACTACTTGC CTTGATTGCT ATCGTCAGGC CCCGCAAGAA GGTAGTACTATGCGGAGACC 2520 CCATGCAATG CGGATTCTTC AACATGATGC AACTAAAGGT ACATTTCAATCACCCTGAAA 2580 AAGACATATG CACCAAGACA TTCTACAAGT ATATCTCCCG GCGTTGCACACAGCCAGTTA 2640 CAGCTATTGT ATCGACACTG CATTACGATG GAAAGATGAA AACCACGAACCCGTGCAAGA 2700 AGAACATTGA AATCGATATT ACAGGGGCCA CAAAGCCGAA GCCAGGGGATATCATCCTGA 2760 CATGTTTCCG CGGGTGGGTT AAGCAATTGC AAATCGACTA TCCCGGACATGAAGTAATGA 2820 CAGCCGCGGC CTCACAAGGG CTAACCAGAA AAGGAGTGTA TGCCGTCCGGCAAAAAGTCA 2880 ATGAAAACCC ACTGTACGCG ATCACATCAG AGCATGTGAA CGTGTTGCTCACCCGCACTG 2940 AGGACAGGCT AGTGTGGAAA ACCTTGCAGG GCGACCCATG GATTAAGCAGCCCACTAACA 3000 TACCTAAAGG AAACTTTCAG GCTACTATAG AGGACTGGGA AGCTGAACACAAGGGAATAA 3060 TTGCTGCAAT AAACAGCCCC ACTCCCCGTG CCAATCCGTT CAGCTGCAAGACCAACGTTT 3120 GCTGGGCGAA AGCATTGGAA CCGATACTAG CCACGGCCGG TATCGTACTTACCGGTTGCC 3180 AGTGGAGCGA ACTGTTCCCA CAGTTTGCGG ATGACAAACC ACATTCGGCCATTTACGCCT 3240 TAGACGTAAT TTGCATTAAG TTTTTCGGCA TGGACTTGAC AAGCGGACTGTTTTCTAAAC 3300 AGAGCATCCC ACTAACGTAC CATCCCGCCG ATTCAGCGAG GCCGGTAGCTCATTGGGACA 3360 ACAGCCCAGG AACCCGCAAG TATGGGTACG ATCACGCCAT TGCCGCCGAACTCTCCCGTA 3420 GATTTCCGGT GTTCCAGCTA GCTGGGAAGG GCACACAACT TGATTTGCAGACGGGGAGAA 3480 CCAGAGTTAT CTCTGCACAG CATAACCTGG TCCCGGTGAA CCGCAATCTTCCTCACGCCT 3540 TAGTCCCCGA GTACAAGGAG AAGCAACCCG GCCCGGTCAA AAAATTCTTGAACCAGTTCA 3600 AACACCACTC AGTACTTGTG GTATCAGAGG AAAAAATTGA AGCTCCCCGTAAGAGAATCG 3660 AATGGATCGC CCCGATTGGC ATAGCCGGTG CAGATAAGAA CTACAACCTGGCTTTCGGGT 3720 TTCCGCCGCA GGCACGGTAC GACCTGGTGT TCATCAACAT TGGAACTAAATACAGAAACC 3780 ACCACTTTCA GCAGTGCGAA GACCATGCGG CGACCTTAAA AACCCTTTCGCGTTCGGCCC 3840 TGAATTGCCT TAACCCAGGA GGCACCCTCG TGGTGAAGTC CTATGGCTACGCCGACCGCA 3900 ACAGTGAGGA CGTAGTCACC GCTCTTGCCA GAAAGTTTGT CAGGGTGTCTGCAGCGAGAC 3960 CAGATTGTGT CTCAAGCAAT ACAGAAATGT ACCTGATTTT CCGACAACTAGACAACAGCC 4020 GTACACGGCA ATTCACCCCG CACCATCTGA ATTGCGTGAT TTCGTCCGTGTATGAGGGTA 4080 CAAGAGATGG AGTTGGAGCC GCGCCGTCAT ACCGCACCAA AAGGGAGAATATTGCTGACT 4140 GTCAAGAGGA AGCAGTTGTC AACGCAGCCA ATCCGCTGGG TAGACCAGGCGAAGGAGTCT 4200 GCCGTGCCAT CTATAAACGT TGGCCGACCA GTTTTACCGA TTCAGCCACGGAGACAGGCA 4260 CCGCAAGAAT GACTGTGTGC CTAGGAAAGA AAGTGATCCA CGCGGTCGGCCCTGATTTCC 4320 GGAAGCACCC AGAAGCAGAA GCCTTGAAAT TGCTACAAAA CGCCTACCATGCAGTGGCAG 4380 ACTTAGTAAA TGAACATAAC ATCAAGTCTG TCGCCATTCC ACTGCTATCTACAGGCATTT 4440 ACGCAGCCGG AAAAGACCGC CTTGAAGTAT CACTTAACTG CTTGACAACCGCGCTAGACA 4500 GAACTGACGC GGACGTAACC ATCTATTGCC TGGATAAGAA GTGGAAGGAAAGAATCGACG 4560 CGGCACTCCA ACTTAAGGAG TCTGTAACAG AGCTGAAGGA TGAAGATATGGAGATCGACG 4620 ATGAGTTAGT ATGGATCCAT CCAGACAGTT GCTTGAAGGG AAGAAAGGGATTCAGTACTA 4680 CAAAAGGAAA ATTGTATTCG TACTTCGAAG GCACCAAATT CCATCAAGCAGCAAAAGACA 4740 TGGCGGAGAT AAAGGTCCTG TTCCCTAATG ACCAGGAAAG TAATGAACAACTGTGTGCCT 4800 ACATATTGGG TGAGACCATG GAAGCAATCC GCGAAAAGTG CCCGGTCGACCATAACCCGT 4860 CGTCTAGCCC GCCCAAAACG TTGCCGTGCC TTTGCATGTA TGCCATGACGCCAGAAAGGG 4920 TCCACAGACT TAGAAGCAAT AACGTCAAAG AAGTTACAGT ATGCTCCTCCACCCCCCTTC 4980 CTAAGCACAA AATTAAGAAT GTTCAGAAGG TTCAGTGCAC GAAAGTAGTCCTGTTTAATC 5040 CGCACACTCC CGCATTCGTT CCCGCCCGTA AGTACATAGA AGTGCCAGAACAGCCTACCG 5100 CTCCTCCTGC ACAGGCCGAG GAGGCCCCCG AAGTTGTAGC GACACCGTCACCATCTACAG 5160 CTGATAACAC CTCGCTTGAT GTCACAGACA TCTCACTGGA TATGGATGACAGTAGCGAAG 5220 GCTCACTTTT TTCGAGCTTT AGCGGATCGG ACAACTCTAT TACTAGTATGGACAGTTGGT 5280 CGTCAGGACC TAGTTCACTA GAGATAGTAG ACCGAAGGCA GGTGGTGGTGGCTGACGTTC 5340 ATGCCGTCCA AGAGCCTGCC CCTATTCCAC CGCCAAGGCT AAAGAAGATGGCCCGCCTGG 5400 CAGCGGCAAG AAAAGAGCCC ACTCCACCGG CAAGCAATAG CTCTGAGTCCCTCCACCTCT 5460 CTTTTGGTGG GGTATCCATG TCCCTCGGAT CAATTTTCGA CGGAGAGACGGCCCGCCAGG 5520 CAGCGGTACA ACCCCTGGCA ACAGGCCCCA CGGATGTGCC TATGTCTTTCGGATCGTTTT 5580 CCGACGGAGA GATTGATGAG CTGAGCCGCA GAGTAACTGA GTCCGAACCCGTCCTGTTTG 5640 GATCATTTGA ACCGGGCGAA GTGAACTCAA TTATATCGTC CCGATCAGCCGTATCTTTTC 5700 CACTACGCAA GCAGAGACGT AGACGCAGGA GCAGGAGGAC TGAATACTGACTAACCGGGG 5760 TAGGTGGGTA CATATTTTCG ACGGACACAG GCCCTGGGCA CTTGCAAAAGAAGTCCGTTC 5820 TGCAGAACCA GCTTACAGAA CCGACCTTGG AGCGCAATGT CCTGGAAAGAATTCATGCCC 5880 CGGTGCTCGA CACGTCGAAA GAGGAACAAC TCAAACTCAG GTACCAGATGATGCCCACCG 5940 AAGCCAACAA AAGTAGGTAC CAGTCTCGTA AAGTAGAAAA TCAGAAAGCCATAACCACTG 6000 AGCGACTACT GTCAGGACTA CGACTGTATA ACTCTGCCAC AGATCAGCCAGAATGCTATA 6060 AGATCACCTA TCCGAAACCA TTGTACTCCA GTAGCGTACC GGCGAACTACTCCGATCCAC 6120 AGTTCGCTGT AGCTGTCTGT AACAACTATC TGCATGAGAA CTATCCGACAGTAGCATCTT 6180 ATCAGATTAC TGACGAGTAC GATGCTTACT TGGATATGGT AGACGGGACAGTCGCCTGCC 6240 TGGATACTGC AACCTTCTGC CCCGCTAAGC TTAGAAGTTA CCCGAAAAAACATGAGTATA 6300 GAGCCCCGAA TATCCGCAGT GCGGTTCCAT CAGCGATGCA GAACACGCTACAAAATGTGC 6360 TCATTGCCGC AACTAAAAGA AATTGCAACG TCACGCAGAT GCGTGAACTGCCAACACTGG 6420 ACTCAGCGAC ATTCAATGTC GAATGCTTTC GAAAATATGC ATGTAATGACGAGTATTGGG 6480 AGGAGTTCGC TCGGAAGCCA ATTAGGATTA CCACTGAGTT TGTCACCGCATATGTAGCTA 6540 GACTGAAAGG CCCTAAGGCC GCCGCACTAT TTGCAAAGAC GTATAATTTGGTCCCATTGC 6600 AAGAAGTGCC TATGGATAGA TTCGTCATGG ACATGAAAAG AGACGTGAAAGTTACACCAG 6660 GCACGAAACA CACAGAAGAA AGACCGAAAG TACAAGTGAT ACAAGCCGCAGAACCCCTGG 6720 CGACTGCTTA CTTATGCGGG ATTCACCGGG AATTAGTGCG TAGGCTTACGGCCGTCTTGC 6780 TTCCAAACAT TCACACGCTT TTTGACATGT CGGCGGAGGA TTTTGATGCAATCATAGCAG 6840 AACACTTCAA GCAAGGCGAC CCGGTACTGG AGACGGATAT CGCATCATTCGACAAAAGCC 6900 AAGACGACGC TATGGCGTTA ACCGGTCTGA TGATCTTGGA GGACCTGGGTGTGGATCAAC 6960 CACTACTCGA CTTGATCGAG TGCGCCTTTG GAGAAATATC ATCCACCCATCTACCTACGG 7020 GTACTCGTTT TAAATTCGGG GCGATGATGA AATCCGGAAT GTTCCTCACACTTTTTGTCA 7080 ACACAGTTTT GAATGTCGTT ATCGCCAGCA GAGTACTAGA AGAGCGGCTTAAAACGTCCA 7140 GATGTGCAGC GTTCATTGGC GACGACAACA TCATACATGG AGTAGTATCTGACAAAGAAA 7200 TGGCTGAGAG GTGCGCCACC TGGCTCAACA TGGAGGTTAA GATCATCGACGCAGTCATCG 7260 GTGAGAGACC ACCTTACTTC TGCGGCGGAT TTATCTTGCA AGATTCGGTTACTTCCACAG 7320 CGTGCCGCGT GGCGGATCCC CTGAAAAGGC TGTTTAAGTT GGGTAAACCGCTCCCAGCCG 7380 ACGACGAGCA AGACGAAGAC AGAAGACGCG CTCTGCTAGA TGAAACAAAGGCGTGGTTTA 7440 GAGTAGGTAT AACAGGCACT TTAGCAGTGG CCGTGACGAC CCGGTATGAGGTAGACAATA 7500 TTACACCTGT CCTACTGGCA TTGAGAACTT TTGCCCAGAG CAAAAGAGCATTCCAAGCCA 7560 TCAGAGGGGA AATAAAGCAT CTCTACGGTG GTCCTAAATA GTCAGCATAGTACATTTCAT 7620 CTGACTAATA CTACAACACC ACCACCATGA ATAGAGGATT CTTTAACATGCTCGGCCGCC 7680 GCCCCTTCCC GGCCCCCACT GCCATGTGGA GGCCGCGGAG AAGGAGGCAGGCGGCCCCGA 7740 TGCCTGCCCG CAACGGGCTG GCTTCTCAAA TCCAGCAACT GACCACAGCCGTCAGTGCCC 7800 TAGTCATTGG ACAGGCAACT AGACCTCAAC CCCCACGTCC ACGCCCGCCACCGCGCCAGA 7860 AGAAGCAGGC GCCCAAGCAA CCACCGAAGC CGAAGAAACC AAAAACGCAGGAGAAGAAGA 7920 AGAAGCAACC TGCAAAACCC AAACCCGGAA AGAGACAGCG CATGGCACTTAAGTTGGAGG 7980 CCGACAGATT GTTCGACGTC AAGAACGAGG ACGGAGATGT CATCGGGCACGCACTGGCCA 8040 TGGAAGGAAA GGTAATGAAA CCTCTGCACG TGAAAGGAAC CATCGACCACCCTGTGCTAT 8100 CAAAGCTCAA ATTTACCAAG TCGTCAGCAT ACGACATGGA GTTCGCACAGTTGCCAGTCA 8160 ACATGAGAAG TGAGGCATTC ACCTACACCA GTGAACACCC CGAAGGATTCTATAACTGGC 8220 ACCACGGAGC GGTGCAGTAT AGTGGAGGTA GATTTACCAT CCCTCGCGGAGTAGGAGGCA 8280 GAGGAGACAG CGGTCGTCCG ATCATGGATA ACTCCGGTCG GGTTGTCGCGATAGTCCTCG 8340 GTGGCGCTGA TGAAGGAACA CGAACTGCCC TTTCGGTCGT CACCTGGAATAGTAAAGGGA 8400 AGACAATTAA GACGACCCCG GAAGGGACAG AAGAGTGGTC CGCAGCACCACTGGTCACGG 8460 CAATGTGTTT GCTCGGAAAT GTGAGCTTCC CATGCGACCG CCCGCCCACATGCTATACCC 8520 GCGAACCTTC CAGAGCCCTC GACATCCTTG AAGAGAACGT GAACCATGAGGCCTACGATA 8580 CCCTGCTCAA TGCCATATTG CGGTGCGGAT CGTCTGGCAG AAGCAAAAGAAGCGTCATTG 8640 ACGACTTTAC CCTGACCAGC CCCTACTTGG GCACATGCTC GTACTGCCACCATACTGTAC 8700 CGTGCTTCAG CCCTGTTAAG ATCGAGCAGG TCTGGGACGA AGCGGACGATAACACCATAC 8760 GCATACAGAC TTCCGCCCAG TTTGGATACG ACCAAAGCGG AGCAGCAAGCGCAAACAAGT 8820 ACCGCTACAT GTCGCTTAAG CAGGATCACA CCGTTAAAGA AGGCACCATGGATGACATCA 8880 AGATTAGCAC CTCAGGACCG TGTAGAAGGC TTAGCTACAA AGGATACTTTCTCCTCGCAA 8940 AATGCCCTCC AGGGGACAGC GTAACGGTTA GCATAGTGAG TAGCAACTCAGCAACGTCAT 9000 GTACACTGGC CCGCAAGATA AAACCAAAAT TCGTGGGACG GGAAAAATATGATCTACCTC 9060 CCGTTCACGG TAAAAAAATT CCTTGCACAG TGTACGACCG TCTGAAAGAAACAACTGCAG 9120 GCTACATCAC TATGCACAGG CCGAGACCGC ACGCTTATAC ATCCTACCTGGAAGAATCAT 9180 CAGGGAAAGT TTACGCAAAG CCGCCATCTG GGAAGAACAT TACGTATGAGTGCAAGTGCG 9240 GCGACTACAA GACCGGAACC GTTTCGACCC GCACCGAAAT CACTGGTTGCACCGCCATCA 9300 AGCAGTGCGT CGCCTATAAG AGCGACCAAA CGAAGTGGGT CTTCAACTCACCGGACTTGA 9360 TCAGACATGA CGACCACACG GCCCAAGGGA AATTGCATTT GCCTTTCAAGTTGATCCCGA 9420 GTACCTGCAT GGTCCCTGTT GCCCACGCGC CGAATGTAAT ACATGGCTTTAAACACATCA 9480 GCCTCCAATT AGATACAGAC CACTTGACAT TGCTCACCAC CAGGAGACTAGGGGCAAACC 9540 CGGAACCAAC CACTGAATGG ATCGTCGGAA AGACGGTCAG AAACTTCACCGTCGACCGAG 9600 ATGGCCTGGA ATACATATGG GGAAATCATG AGCCAGTGAG GGTCTATGCCCAAGAGTCAG 9660 CACCAGGAGA CCCTCACGGA TGGCCACACG AAATAGTACA GCATTACTACCATCGCCATC 9720 CTGTGTACAC CATCTTAGCC GTCGCATCAG CTACCGTGGC GATGATGATTGGCGTAACTG 9780 TTGCAGTGTT ATGTGCCTGT AAAGCGCGCC GTGAGTGCCT GACGCCATACGCCCTGGCCC 9840 CAAACGCCGT AATCCCAACT TCGCTGGCAC TCTTGTGCTG CGTTAGGTCGGCCAATGCTG 9900 AAACGTTCAC CGAGACCATG AGTTACTTGT GGTCGAACAG TCAGCCGTTCTTCTGGGTCC 9960 AGTTGTGCAT ACCTTTGGCC GCTTTCATCG TTCTAATGCG CTGCTGCTCCTGCTGCCTGC 10020 CTTTTTTAGT GGTTGCCGGC GCCTACCTGG CGAAGGTAGA CGCCTACGAACATGCGACCA 10080 CTGTTCCAAA TGTGCCACAG ATACCGTATA AGGCACTTGT TGAAAGGGCAGGGTATGCCC 10140 CGCTCAATTT GGAGATCACT GTCATGTCCT CGGAGGTTTT GCCTTCCACCAACCAAGAGT 10200 ACATTACCTG CAAATTCACC ACTGTGGTCC CCTCCCCAAA AATCAAATGCTGCGGCTCCT 10260 TGGAATGTCA GCCGGCCGCT CATGCAGACT ATACCTGCAA GGTCTTCGGAGGGGTCTACC 10320 CCTTTATGTG GGGAGGAGCG CAATGTTTTT GCGACAGTGA GAACAGCCAGATGAGTGAGG 10380 CGTACGTCGA ATTGTCAGCA GATTGCGCGT CTGACCACGC GCAGGCGATTAAGGTGCACA 10440 CTGCCGCGAT GAAAGTAGGA CTGCGTATTG TGTACGGGAA CACTACCAGTTTCCTAGATG 10500 TGTACGTGAA CGGAGTCACA CCAGGAACGT CTAAAGACTT GAAAGTCATAGCTGGACCAA 10560 TTTCAGCATC GTTTACGCCA TTCGATCATA AGGTCGTTAT CCATCGCGGCCTGGTGTACA 10620 ACTATGACTT CCCGGAATAT GGAGCGATGA AACCAGGAGC GTTTGGAGACATTCAAGCTA 10680 CCTCCTTGAC TAGCAAGGAT CTCATCGCCA GCACAGACAT TAGGCTACTCAAGCCTTCCG 10740 CCAAGAACGT GCATGTCCCG TACACGCAGG CCTCATCAGG ATTTGAGATGTGGAAAAAGA 10800 ACTCAGGCCG CCCACTGCAG GAAACCGCAC CTTTCGGGTG TAAGATTGCAGTAAATCCGC 10860 TCCGAGCGGT GGACTGTTCA TACGGGAACA TTCCCATTTC TATTGACATCCCGAACGCTG 10920 CCTTTATCAG GACATCAGAT GCACCACTGG TCTCAACAGT CAAATGTGAAGTCAGTGAGT 10980 GCACTTATTC AGCAGACTTC GGCGGGATGG CCACCCTGCA GTATGTATCCGACCGCGAAG 11040 GTCAATGCCC CGTACATTCG CATTCGAGCA CAGCAACTCT CCAAGAGTCGACAGTACATG 11100 TCCTGGAGAA AGGAGCGGTG ACAGTACACT TTAGCACCGC GAGTCCACAGGCGAACTTTA 11160 TCGTATCGCT GTGTGGGAAG AAGACAACAT GCAATGCAGA ATGTAAACCACCAGCTGACC 11220 ATATCGTGAG CACCCCGCAC AAAAATGACC AAGAATTTCA AGCCGCCATCTCAAAAACAT 11280 CATGGAGTTG GCTGTTTGCC CTTTTCGGCG GCGCCTCGTC GCTATTAATTATAGGACTTA 11340 TGATTTTTGC TTGCAGCATG ATGCTGACTA GCACACGAAG ATGACCGCTACGCCCCAATG 11400 ATCCGACCAG CAAAACTCGA TGTACTTCCG AGGAACTGAT GTGCATAATGCATCAGGCTG 11460 GTACATTAGA TCCCCGCTTA CCGCGGGCAA TATAGCAACA CTAAAAACTCGATGTACTTC 11520 CGAGGAAGCG CAGTGCATAA TGCTGCGCAG TGTTGCCACA TAACCACTATATTAACCATT 11580 TATCTAGCGG ACGCCAAAAA CTCAATGTAT TTCTGAGGAA GCGTGGTGCATAATGCCACG 11640 CAGCGTCTGC ATAACTTTTA TTATTTCTTT TATTAATCAA CAAAATTTTGTTTTTAACAT 11700 TTCAAAAAAA AAAAAAAAAA AAAAAAAAAA AAAAAAAAAA 11740 6amino acids amino acid single linear 104 Ser Ile Leu Gly Ser Arg 1 5 21base pairs nucleic acid single linear 105 GTCCGTTTGT CGTGCAACTG C 21 21base pairs nucleic acid single linear 106 GTCCGTTTGT CGTGCAACTG A 21 21base pairs nucleic acid single linear 107 CAATCTTCCT CACGCCTTAG C 21 21base pairs nucleic acid single linear 108 CAATCTTCCT CACGCCTTAG T 21 21base pairs nucleic acid single linear 109 TCCTAAATAG TCAGCATAGT A 21 21base pairs nucleic acid single linear 110 TCCTAAATAG TCAGCATAGT T 21 34base pairs nucleic acid single linear 111 TATATCTCGA GGGTGGTGTTGTAGTATTAG TCAG 34 35 base pairs nucleic acid single linear 112TATATGATAT CAAAAAGCCT GAACTCACCG CGACG 35 35 base pairs nucleic acidsingle linear 113 ATATAGGATC CTCAGTTAGC CTCCCCCATC TCCCG 35 120 aminoacids amino acid <Unknown> linear 114 Met Asn Tyr Ile Pro Thr Gln ThrPhe Tyr Gly Arg Arg Trp Arg Pro 1 5 10 15 Arg Pro Ala Phe Arg Pro TrpGln Val Ser Met Gln Pro Thr Pro Thr 20 25 30 Met Val Thr Pro Met Leu GlnAla Pro Asp Leu Gln Ala Gln Gln Met 35 40 45 Gln Gln Leu Ile Ser Ala ValSer Ala Leu Thr Thr Lys Gln Asn Val 50 55 60 Lys Ala Pro Lys Gly Gln ArgGln Lys Lys Gln Gln Lys Pro Lys Glu 65 70 75 80 Lys Lys Glu Asn Gln LysLys Lys Pro Thr Gln Lys Lys Lys Gln Gln 85 90 95 Gln Lys Pro Lys Pro GlnAla Lys Lys Lys Lys Pro Gly Arg Arg Glu 100 105 110 Arg Met Cys Met LysIle Glu Asn 115 120 103 amino acids amino acid <Unknown> linear 115 MetAsn Tyr Ile Pro Thr Gln Thr Phe Tyr Gly Arg Arg Trp Arg Pro 1 5 10 15Arg Pro Ala Phe Arg Pro Trp Gln Val Ser Met Gln Pro Thr Pro Thr 20 25 30Met Val Thr Pro Met Leu Gln Ala Pro Asp Leu Gln Ala Gln Gln Met 35 40 45Gln Gln Leu Ile Ser Ala Val Ser Ala Leu Thr Thr Lys Gln Asn Val 50 55 60Lys Ala Pro Lys Gly Gln Arg Gln Lys Lys Gln Gln Lys Pro Lys Glu 65 70 7580 Lys Lys Glu Asn Gln Lys Lys Lys Pro Thr Leu Lys Arg Arg Glu Arg 85 9095 Met Cys Met Lys Ile Glu Asn 100 88 amino acids amino acid <Unknown>linear 116 Met Asn Tyr Ile Pro Thr Gln Thr Phe Tyr Gly Arg Arg Trp ArgPro 1 5 10 15 Arg Pro Ala Phe Arg Pro Trp Gln Val Ser Met Gln Pro ThrPro Thr 20 25 30 Met Val Thr Pro Met Leu Gln Ala Pro Asp Leu Gln Ala GlnGln Met 35 40 45 Gln Gln Leu Ile Ser Ala Val Ser Ala Leu Thr Thr Lys GlnAsn Val 50 55 60 Lys Ala Pro Lys Gly Gln Arg Gln Lys Lys Gln Leu Lys ArgArg Glu 65 70 75 80 Arg Met Cys Met Lys Ile Glu Asn 85 76 amino acidsamino acid <Unknown> linear 117 Met Asn Tyr Ile Pro Thr Gln Thr Phe TyrGly Arg Arg Trp Arg Pro 1 5 10 15 Arg Pro Ala Phe Arg Pro Trp Gln ValSer Met Gln Pro Thr Pro Thr 20 25 30 Met Val Thr Pro Met Leu Gln Ala ProAsp Leu Gln Ala Gln Gln Met 35 40 45 Gln Gln Leu Ile Ser Ala Val Ser AlaLeu Thr Thr Lys Gln Asn Leu 50 55 60 Lys Arg Arg Glu Arg Met Cys Met LysIle Glu Asn 65 70 75 38 base pairs nucleic acid single linear 118ATATAGGATC CTTCGCATGA TTGAACAAGA TGGATTGC 38 5 amino acids amino acid<Unknown> linear 119 Leu Xaa Pro Gly Gly 1 5 6 amino acids amino acid<Unknown> linear 120 Leu Asn Pro Gly Gly Thr 1 5 6 amino acids aminoacid <Unknown> linear 121 Leu Lys Pro Gly Gly Thr 1 5 6 amino acidsamino acid <Unknown> linear 122 Leu Lys Pro Gly Gly Ile 1 5 6 aminoacids amino acid <Unknown> linear 123 Leu Xaa Pro Gly Gly Ser 1 5 6amino acids amino acid <Unknown> linear 124 Leu Asn Thr Gly Gly Thr 1 55 amino acids amino acid <Unknown> linear 125 Leu Xaa Pro Gly Gly 1 5

We claim:
 1. An alphavirus packaging cell line, comprising a host celland a DNA alphavirus structural protein expression cassette, whereinsaid alphavirus structural protein expression cassette comprises a 5′promoter that directs synthesis of an RNA transcript, a nucleic acidsequence encoding at least one alphavirus structural protein and anucleic acid sequence encoding a selectable marker.
 2. The expressioncassette according to claim 1, wherein said alphavirus structuralprotein cassette further comprises a 5′ sequence that initiatestranscription of alphavirus RNA, an alphavirus viral junction promotersequence, and an alphavirus RNA polymerase recognition sequence.
 3. Analphavirus packaging cell comprising a cell and an alphavirus structuralprotein expression cassette, wherein said alphavirus structural proteinexpression cassette comprises: (a) a 5′ promoter that directs synthesisof RNA; (b) a 5′ sequence that initiates transcription of alphavirusRNA; (c) a nucleic acid sequence encoding an alphavirus junction regionpromoter; (d) a nucleic acid sequence encoding one or more alphavirusstructural proteins; (e) a nucleic acid sequence encoding a selectablemarker; and (1) an RNA polymerase recognition sequence.
 4. Thealphavirus packaging cell line according to any one of claims 1, 3, or 2wherein the nucleic acid sequence that encodes a selectable marker ispositioned upstream from the nucleic acid sequence that encodes at leastone alphavirus structural protein.
 5. The alphavirus packaging cell lineaccording to any one of claims 1, 3, or 2 wherein the nucleic acidsequence that encodes a selectable marker is positioned downstream fromthe nucleic acid sequence that encodes at least one alphavirusstructural protein.
 6. The alphavirus packaging cell line according toany one of claims 1, 3, or 2 wherein said 5′ promoter is a eukaryoticpromoter.
 7. The alphavirus packaging cell line according to claim 6wherein said promoter is an RNA polymerase II promoter (pol II).
 8. Thealphavirus packaging cell line according to claim 7 wherein said pol IIpromoter is a cytomegalovirus (CMV) promoter or Rous sarcoma virus (RSV)promoter.
 9. The alphavirus packaging cell line according to claim 6further comprising a 3′ sequence that controls transcriptiontermination.
 10. The alphavirus packaging cell line according to claimany one of claims 1, 3, or 2 wherein said 5′ promoter is an induciblepromoter.
 11. The alphavirus packaging cell line according to any one ofclaims 1, 3, or 2 further comprising a catalytic ribozyme processingsequence.
 12. The alphavirus packaging cell line according to any one ofclaims 1, 3, or 2 further comprising an element that facilitates RNAexport from the nucleus.
 13. The alphavirus packaging cell lineaccording to claim 12 wherein said element that facilitates RNA exportis a hepatitis B virus (HBV) post-transcriptional regulatory element(PRE) sequence.
 14. The alphavirus packaging cell line according to anyone of claims 1, 3, or 2 further comprising an element that permitstranslation of multicistronic mRNA.
 15. The alphavirus packaging cellline according to claim 14 wherein said element that permits translationof multicistronic mRNA is positioned between said nucleic acid sequenceencoding one or more alphavirus structural proteins and said nucleicacid sequence encoding a selectable marker.
 16. The alphavirus packagingcell line according to claim 15 wherein said element that permitstranslation of multicistronic mRNA is an Internal Ribosome Entry Site(IRES).
 17. The alphavirus packaging cell line according to claim 15wherein said element that permits translation of multicistronic mRNA isan element that permits ribosomal read through.
 18. The alphaviruspackaging cell line according to claim 17 wherein said element thatpermits ribosomal read through is a BiP sequence.
 19. The alphaviruspackaging cell line according to claim any one of claims 1, 3, or 2further comprising a sequence that enhances translation.
 20. Thealphavirus packaging cell line according to claim 19 wherein saidsequence that enhances translation is a deleted form of a Ross Rivervirus capsid protein gene.
 21. The alphavirus packaging cell lineaccording to any one of claims 1, 3, or 2 wherein said at least onealphavirus structural protein is a capsid, and wherein said alphavirusstructural protein expression cassette does not express an alphavirusenvelope glycoprotein.
 22. The alphavirus packaging cell line accordingto any one of claims 1, 3, or 2 wherein said at least one alphavirusstructural protein is an envelope glycoprotein, and wherein saidalphavirus structural protein expression cassette does not express analphavirus capsid protein.
 23. The alphavirus packaging cell lineaccording to any one of claims 1, 3, or 2 wherein said nucleic acidsequence encoding a selectable marker further comprises an alphavirusnsP1 nucleic acid sequence wherein said nsP1 sequence is an in-framefusion with said nucleic acid sequence encoding a selectable marker. 24.The alphavirus packaging cell line according to any one of claims 1, 3or 2 further comprising an element that permits ribosomal read throughpositioned between said nucleic acid sequence encoding one or morealphavirus structural proteins and said nucleic acid sequence encoding aselectable marker.
 25. The alphavirus packaging cell line according toclaim 24 wherein said element that permits ribosomal read through is aBiP sequence.
 26. The alphavirus packaging cell line according to claim3 wherein said selectable marker is positioned between a 5′ sequence of(b) and an alphavirus junction region promoter of (c).
 27. Thealphavirus packaging cell line according to claim 3 wherein saidselectable marker is positioned between a nucleic acid sequence of (d)and the polymerase recognition sequence of (f).
 28. The alphaviruspackaging cell line according to any one of claims 1, 3, 2,26,27 whereinsaid selectable marker is encoded by a neomycin resistance gene orhygromycin resistance gene.